Bacterial vectors.

PROCESS FOR PREPARING BACTERIAN VECTORS

Affiliate

The invention relates to the field of genetic engineering and provides novel molecules with recombinant DNA; which comprise multifunctional origins of replication and / or a DNA sequence encoding the most abundant protein in the Lactococcus spec culture supernatant, designated Major Secretion Protein, MSP; its signal peptide and / or MSP gene promoter »New molecules are used for the production of new shuttle vectors for the cloning of DNA in at least E. coli s Lactococcus spsc, or new vectors for the expression of homologous or heterologous genes and for the production of secretion gene products in large positive bacteria such as Lactococcus

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The present invention relates to the field of genetic engineering and provides novel DNA molecules comprising multifunctional origins of replication and / or a DNA sequence encoding the most abundant protein in Lactococcus lactis culture supernatant, hereinafter referred to as the Main Secretion Product. (MSP), its signal peptide and / or the MSP gene promoter. The new DNA molecules were used for the production of new shuttle vectors for the cloning of DNA in at least E. coli and Lactococcus spec, or new vectors for the expression of homologous or heterologous genes and production of Brsm-secreted bacterial products. such as Lactococcus ..... spgç ^, s Bacillus spec ..

h.und.amen, to „, dp Invention

Although in engineering techniques, genetics, numerous prokaryotic vector-host systems for cloning heterologous or homologous genes are already known, there is a continuing need for new systems that may have re-advantageous advantages to systems. known.

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Most of the recombinant work in DNA technology has been done with bacteria such as Escherichia coli or Bacillus subtilis. Lactic acid bacteria, however, are of much more industrial interest. For this reason, efforts have been made to develop plasmid vectors for cloning and expression of homologous and heterologous genes in lactic acid bacteria, particularly in Lacto-bacilli spec spec, and Lactococcus spec. Pus WD Bb / 0394o, Basson and Anderson (1985)₅ EP-A-336, 677, Bates et al. (1989). dos st al., (1985) these review articles Laçtocpççus ..... speç. it was

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formerly known as Streptococcus soec »

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The lactococcal strains so far investigated carry a characteristic plasmid complement consisting of several different plasmids. (This property can be used to differentiate various lactococcal strains (CDavíes et al., 1981)). For genetic studies, strains without plasmids were constructed by repeated deletion in the course of plasmid function studies (De Vos, 1987). '

For example, the L. lactis 712 'plasmid complement hereinafter referred to as the' L 'lactis LL712, consists of 5 plasmids having the molecular weights of 1, B Md, 2.5 Md, 5.2 Md, 9 Md and 33 Md which were designated pSK71, pSH72, pSH73, pSH74 and pLP712, respectively CBasson, 1983) =

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Based on plasmid pSH7í ds L. lactis s in the related plasmid pWvwI of Cremoels COtio et al, 1982) various cloning vectors were constructed. The cloning vectors were produced by inserting genetic markers such as antibiotic resistance genes into the plasmids or by screening for plasmid fragments for the origin of replication function, ie »ability to support replication of selected DNA fragments» A cloning vector produced according to this latter method is pNZ12 which contains the 1.7 kp Cia1 restriction fragment of p8H71 comprising the origin of replication -CSasson and Anderson, 1985). The origin of replication of pSH71 is also functional in other Bram-positive bacteria such as Bacillus spec, and in the Bram-negative Escherichia Coll.

') you

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Based on these plasmids, cloning vectors useful for introducing expression of homologous or heterologous genes into lactic acid bacteria have been developed. 0 development

cloning vectors have resulted in better-transformed transformed lactococcal strains which are useful in the food industry, for example a% t: r strain.

<img file="PT97081B_D0007.tif" />

316 6 / z) scieloepapos or liib-s s? Stir'ps ds? L_O 1 -actis producing bovine procchymosin CPCT WO 85 / ®3945) »The development of cloning vectors for the production of homologous or heterologous gene products is of interest not only for the production of improved lactic acid bacterial cells but also for the production of recombinant proteins. »One of the main problems with the production of heterologous proteins in expression systems is that the purification of intracellular proteins is time consuming and many results in yields

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»; __-, j __„ i ..

(CII) may be useful if the product is secreted by the host cell. To avoid the problems of purification of bacterially expressed products, host-vector systems for producing recombinant proteins which are supernatant secreted may be useful. »Weak weak ft purification

A further advantage of proteins can be that they can have a native and biologically active conformation since then the refolding process is necessary.

Intracellularly deposited iPKutififiu »Secretion of a protein generally requires a signal peptide at the primary translational end that directs the protein to the advantage if the signal peptide is cleaved protein during translocation through this, however» is not always the case »mine of the road product secret »Has enzymatically from memory^- * ana cexu 1 ar »

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It is an object of the invention to provide new

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<td>vas and gr</td><td>negative and / or negative</td><td>naked ©</td><td>per mi. t.</td><td>-3.ΓΠ</td><td></td><td>in</td><td>genes</td>

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<td>insertion</td><td>of DNA that comprises</td><td>L.3 tX)</td><td>rsgxMo</td><td>of</td><td>mp = 100 ° C 3-5.</td><td>sequ</td><td>emphasis</td>

coding DMA, signal peptide and the coding region of a hitherto unknown gene encoding a polypeptide that is the most abundant protein in the L. lactis supernatant as assessed after precipitation with the TDS supernatant supernatant TCA - protein polyacrylamide precipitates and coloring of the Coomassie brilliant blue gel which is referred to herein as the Principal Product Tissue Secretion (MPS) »

As another solution for a purpose, I have found new hybrid vectors comprising a plasmid derived replication origin of 2.5 Md or 0 = 2 Md from a related replication source, which is functional in hacker.

IdLLib i_.i_ / i2 ua positive and prayerful grara »

So »provide new promoter _? The sequence of the coding region or another solution for a purpose of the invention is a hybrid vector comprising the DNA peptide coding region of the signal peptide and / or the MSP gene or a related gene adora, and a action derived from plasmid 2 »5 Md or 5 = 2 Md of L„ a related origin of replication »

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The invention relates to novel DNA inserts or are useful for secretion production of proteins. The invention provides for the production of new DNA molecules and for the production of host products transformed with a pure propylated invention.

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Detailed Description of the Invention

Molecules of the invention relate to a hybrid vector comprising

a.) the EcoR1 / SalI insert of about 3.5 K.bp of

L. lactis of the pUCRS plasmid or a functional fragment thereof or with an • eu. 3 ds DNA which is a naturally hybridizing or hybridizing to or reference to or including reference to and including an insertion region or a region of te linked to such a degenerate sequence of a DNA sequence that is encompassed in a) or b) and which encodes a signal peptide or

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a derivative of a molecule of an encompassed s / or 'ffi -5:? , b) OR C&gt;

e) the origin of replication ds (I) of plasmid 2 '5 Md ds I-v 1 actisLL712 or ds C11') of plasmid 5 = 2 Md ds L 'lactisLL712 or of CHI) a plasmid of the same plasmid incompatibility group 2 = 5 Md or plasmid 5 = 'from L' 1 sc tx sLL / 12 '

Md

The 3.0 rIph Lactis- Ecotyl / S-al I insert of plasmid pUCRS comprises the promoter region of the MSP gene, the DNA sequence encoding the signal peptide of MSP and the coding region of MSP, MSP with its Signal-bound peptide is hereinafter referred to as the pre-MSP. The EcoRI / SalI insert comprises a. 1926 bp long DNA sequence of SEQ ID MEI described in the sequence listing in an orientation such that base position 1 is proximally to the EcoRI site.

The ds Mf'S coding region extends ···

492 to base position 1793 sequence with vEu

ID

N51 a

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The DMA sequence coding for: length ds extends? / Ammonium d signal peptide

<td>prosuotor region</td><td>here d st</td>

<td>DNA encoding the</td><td>MSP mad</td>

<td>to position 491 d</td><td>&amp; S-S'C | LíSó ”j</td>

how cotfu j_ ₈ what _ç Seq IDs ssss from the end of the sequence following de MSP or a functional fragment thereof are the secretariat of a host cell, and lactic acid such as Lactococcus lactis

<td>the position</td><td>ds bses 411</td>

<td>MSI »0 ft</td><td>signal peptide</td>

<td>with what</td><td>a protein</td>

<td>g »of a</td><td>bacterium</td>

<td>or Bac</td><td>illus spec. »</td>

as B thurínqiensis, if the extreme M of. covalently linked to the C-terminus of the C-covalent peptide of the MPS signal peptide protein is signal »

LUTíc- ρΓ * GXjE: XΠ £ GLIS: 'x-tí Ο% ί' 'νΧΠ «4 3. Ή · £ 5Γ * ÍSXC tifera can be obtained by producing a fusion sequence in which the entire coding DNA sequence of the MPS signal peptide or coding portion of the functional fragment of the MPS signal peptide comprising the C-terminus is linked at. reading grid

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dfcf Ui „3. A 5 '-specific correct cell is a structural framework which is a desired protein. Such a fusion gene is, for example, the DNA molecule with StU ID No. 22 which comprises the sequence of an MSP signal peptide coding sequence and the structural qene of hirudin.

The amino acid sequences of the MSP MSF signal peptide are also shown in sequence with SEQ TO N2I =

<td>The region</td><td>of</td><td>prosoto</td><td>r of the MrS gene</td><td>is there tuada -a</td><td>mountain</td>

<td>ts of sequence d &lt;</td><td>_s</td><td>DNA cod</td><td>if icsdors do</td><td>signal peptide</td><td>MSP and</td>

<td>Understands</td><td>here</td><td>130Θ,</td><td>preferably</td><td>went until about</td><td>100 to</td>

<td>1000 nucleotides »</td><td>Us</td><td>in-Brace</td><td>EcoRI / SalI</td><td>HQ SprOX ii &amp; sdsniGn</td><td>3.5</td>

<td>Kbp in length</td><td>in</td><td>L = 1ac t</td><td>plasmid ions</td><td colspan="2">deo pUCKS, the region of</td>

—From the end of the cut insert: the promoter extends the range of Cf NA with par ticant, restrainting position 1 to. The tor position can bind to RNA polymerase and can control the expression d # te of the base corresponding to è. position 1 of sequence SEQ ID NQl through è. base position 411 »Plus promoter region extends from first

So HindiII situated upstream of the base corresponds to bases 411 = The promoassim region as regulatory proteins is a structural gene operably linked thereto containing the promoter.

A term functional fragment includes fragments d? signal and / or structural functions 'contains' the

Preferred functional fragments are those region of the MSP promoter, the sequence of which nwA

<img file="PT97081B_D0015.tif" />

ds MPS signal peptide coding or promoter region and DMA sequence ds MF signal peptide coding * 3. Fragments according to the invention are also comprised of smaller fragments retaining MSP promoter and / or coding activity of a peptide with signal peptide activity »

Promoter-function fragments, for example, those starting with the first base at the EcoRI cut-off end of the approximately 3.5 kbp L-lactis EcoRI / SalI insert or in particular those starting at the HindIII site and extending to a base corresponding to the approximate position 410 of the SEQ ID MSI sequence »Other ds -promotor-function fragments were selected from the group of fragments starting with either base between the EcoRI site and the HindiII site and ending with a base around position 410 of this sequence. SEQ ID NO: D = smaller fragments of the promoter region also retain promoter activity; a DMA fragment encoding a signal peptide extends for example from approximately base 411 to base 491 of the sequence with SEQ ID IMS! may extend at the 5 'end with a promoter region fragment that does not retain promoter activity.'

A fragment retaining the function of MSP promoter and encoding a signal peptide extends from the EcoRI-cut end of the insert upstream of the base corresponding to position 1 of the DNA sequence of SEQ ID NO: 21 to base position 491. another fragment retaining MSP promoter function encoding a signal peptide extends from the first HindIII restriction site upstream of the base corresponding to position 1 of the DNA sequence with SEQ ID.

<img file="PT97081B_D0016.tif" />

Até21 to around base corresponding to position 491. Other fragments retaining the function of MSP promoter and encoding a signal peptide were selected from the group of fragments starting with either base between said EcoRI and Hindi sítios sites and ending on a base by. around position 491 of the sequence with SEQ ID N21 »

Fragments may contain adapters that provide binding to other DMA molecules. Suitable adapters for the above fragments have a DNA sequence that conforms to a DNA restriction site to which the fragment will bind. They may also contain a DNA site. of predetermined restriction »

A DNA molecule which hybridizes to said insert is hybridized under conventional conditions. A conventional hybridization process is described e.g. by Benton and Davis C1977. Such a hybridizing DNA molecule comprises for example a hybridizing DNA sequence. MSP-encoding structural gene variant. Such hybridization variant is hereinafter also referred to as a structural gene related to the MSP gene. The protein encoded by such related gene is referred to. as a MSP related protein. Thus a promoter region that is naturally bound to. a DNA sequence that hybridizes to said insert is a promoter region of a gene related to the MPS gene »

A structural gene related to the MPS gene is for example a natural variant derived from another bacterium other than L. lactis, particularly from another lactic acid bacterium for example Laçtobaçillus_spec4. or Lactococçus spec »„ e »g» L »cremoris or L» thermophilus »is also a natural variant which is encoded by an isochene on the L. lactis chromosome or on a plasmid that exists naturally on L» lactis »

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The DMA molecules of the invent ι 5 ·· -; Π i ΛιΤ «5? Σ ^ ιΤί ~ ΐΓίΗ £ Ξ-- 1/5“.

which have degenerate DMA sequences are not within the meaning of the genetic code in that an unlimited number of nucleotides are substituted by other nucleotides without altering the amino acid sequence they encode. Molecules having such degenerate DMA sequences may be useful because of their different restriction sites or because of a preferred codon usage in a particular host. Preferred are the degenerate sequences of the DMA sequence encoding the MSP signal peptide or a variant thereof.

molecule in relation to an o · or c / includes fragments, such a term DMA molecule, D

The term derivative when used DMA encompassed in a), mutants or larger derivative derivatives also includes larger fragments or mutant derivatives of such a DNA molecule. Preferred are DNA fragments that retain promoter, signal or structural functions. Examples of such fragments are given below.

A mutant ds a DMA molecule described in a), b) or

c) is for example a DMA molecule having a point deletion, insertion, inversion or mutation that may occur naturally or be artificially introduced into the DMA molecule in vivo or in vitro according to conventional methods. A mutant may have an altered restriction pattern,

Larger derivatives of an isle-encompassed DNA molecule can be found in a L, lactis genomic library.

a), b) or c) are for example removable from the genome of Li

<img file="PT97081B_D0018.tif" />

LM023 © obtained by nucleic acid fragmentation, treatment of the fragments with a suitable restriction enzyme, eg Sau3AI, the

<td>tcoRJ.,</td><td>BamH I</td><td>or</td><td>Hindi11₅ binding in an ad vector</td><td>equado hey</td>

<td>1ambda</td><td>EMBL3</td><td>OR</td><td>plasmid oBR322, clonapem</td><td>AND^: u G ç? íi £</td>

<td>nova r</td><td>£ 5 ITLE</td><td>with</td><td>the same or another re enzyme</td><td>addiction</td>

Larger derivatives of a DNA molecule encompassed in a), b) or c) are also recombinant DMA molecules with flanking sequences, for example those comprising adapters providing suitable restriction sites or placing

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used in constructing a hybrid vector »Flanaue sequences previously contained in the larger derivatives may give rise to fusion genes.

An origin of r

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or start

5 = 2 Md of L. lactis LL712 or of plasmid osipatibility 2.5 and r with which a plasmid repeats

<td>.. Life</td><td>sri</td><td>of plasmid 2.5</td><td>Md</td>

<td>one</td><td>pl</td><td>asmid in the same group</td><td>in</td>

<td>Md</td><td>GU</td><td>of plasmid 5.2 Md pc</td><td>-? U izf</td>

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1 in 1 at_ Lu or L „cremoris and different from E» ocus,

eg

"lactis, in E. coli or lactic acid bacteria" or also in bacteria:

gram negative bacteria: m positive different in Bacillus spec. such as

An origin of replica or 5.2 Md of L. lactis LL.712 or plasmid mismatch for example contained in the total obtained after treatment with which it aerosizes a plasmid of the same group as ds in the 12.2 Md or plasmid 5.2 is respective linearized plasmid endonucleas-s Proper restriction = Thus a hybrid vector of the invention is also one which comprises the entire DNA sequence of plasmid 2 = 5 Md or 5 = 2 Md or a plasmid of the same plasmid mismatch group 2 = 5 Md or 5 = 2 Md »Both Plasmids may be isolated, for example, from strain L1 lacfcisLL.712 which is filed with DSM as DSM 58Ô4.

A DMA sequence that functions as a replication source is also a fragment of any of said plasmids retaining the origin of replication function. Such a DNA fragment can be obtained, for example, by isolation after fragmentation of the respective plasmid or fragment thereof. for example with physical forces, eg tangential forces or with chemical cleavage reactions or with DNA cleaving enzymes such as nucleases, and the Bal-3i exonucleases, Si or exonuclease III or endonucleases, and = g = restriction endonucleases. In particular, such a DNA fragment is a restriction fragment obtained after treatment of the respective plasmid or fragment thereof with one or two different restriction endonucleases which are in the case of plasmid 2 = 5 Md-_; for example Ndel, Sph1 and / or EcoRV and in the case of plasmid 5 = 2 Md, for example Sau3ft, Ndel, HindIII, Accl and / or EcoRI.

The repeat origin of plasmid 2 = 5 Md is. situated for example in the approximately 1.8 kbp Ndel / Sphl fragment in the approximately 3.1 kbp EcoRV / Sphl fragment obtained by partial EcoRV digestion of the approximately 3.5 kbp Sphl fragment and the Sphl fragment. approximately 3,5 Kbp in length, the approximately 1.2 kbp Ndel / EcoRV fragment, the approximately 2.5 kbp EcoRV fragment or the approximately 2.5 kbp EcoRV / Sphl fragment obtained after partial EcoRV digestion of the approximately Sphl fragment 3.5 Kbp »D Sphl fragment approximately 3.5 Kbp in length and other fragments referred to herein are also included in pSC12 = Production of pSC12 is described in Example 1.2 = A map is shown in Figure 2 of restriction »

The origin of replication of the 5.2 Md plasmid is located for example in the approximately 1.2 Kbp Mdel / HindIII fragment, in the approximately 2.2 Kbp Ndel / Accl fragment in the approximately 2 Ndel / EcoRI fragment. , 7 Kbp, the approximately 3.1 Kbp long NdeI / Sau3A fragment in the approximately 2.2 Kbb long Sau3AzHindiII fragment in the approximately 3.2 Kbp long Sau3A / AccI fragment, SauA / EcoRI fragment approximately 3.7 Kbp in length or Sau3A fragment of about 4 = 1 Kbp in length. Fragments with the Ssu3A ends were obtained after partial digestion with Sau3A of plasmid 5.2Md, ds pSCIS or fragments. restriction constraints of these plasmids. pSCIS production is described in Example 3. »Figure 3 shows a restriction map.

Plasmids of the same incompatibility group cannot stably coexist in it; cell »They carry related replication sources»

A recombinant DNA molecule contains for example adapters and / or sequences derived from a vector such as a phage or plasmid and optionally a marker gene such as a resistance marker gene, and&gt; g = an erythromycin, ampicillin or tetracycline or the like. A recombinant DNA molecule comprising an origin of replication of the present invention is for example a lactic acid bacteria plasmid vector, preferably a shuttle vector that can replicate at least in lactic acid bacteria and E.

<img file="PT97081B_D0021.tif" />

coli and wherein a single diocholic origin derived from the actococci to the invention is^-) j_x. „R ·» poUiXulíi t.cai ”» 1

A preferred shuttle vector is pSC12deltaN, p5C12dsltaP, pSC12deI taiMP, pSClS, pSCIBdeltaP or pSCisdeltaN. A preferred shuttle vector for the expression of a foreign gene in lactic acid bacteria is pSC12HIRR-1 or pSC12HIR. Preferred examples are described in the Examples.

A sequence of ϋ-ΝΑ that fuses ion to a replication is at least in the lactic acid bacteria and smfc.

intended for inclusion in e) is also a point inversion, insertion, deletion or mutation of a DMA sequence already defined herein which retains the origin function of replication 'such mutants have an altered nucleotide sequence in the sequences of) IMA f 1 antisense repeats of actual replication 'by point mutations that cause an altered restriction pattern or deletion mutants such as the L insertions. lactis of plasmids pSC12deltaP, pSC12deltaN, pSC12deltaNP, pSCISdeltaP or pSCiSdeltaN (see Table 1). The actual source of replication referred to above is the smallest DMA fragment that acts as source □ &lt;

application"

Hybrid vectors of cloning into hosts such as fungi or particular bacteria. They may be derived from any vector useful in genetic engineering, such as phages, cosmids or plasmids. They are, for example, derived from lambda phage, e.g. 39939 or EMBL3, thio phage Mi3 »e = g» Hi-3mpi or M3mpi9 of bacterial plasmids, eg »pBR 322, pUCIS or pUC19 or lactic acid bacterial plasmids or yeast plasmids, eg» yeast 2 μm plasmid or also the disruptive phages or disruptive plasmid in the

the presence of an helper phage or helper plasmid allowing replication of said defective phages or plasmids.

<img file="PT97081B_D0022.tif" />

<img file="PT97081B_D0023.tif" />

A hybrid vector of the invention comprises a nucleotide sequence of a DNA molecule encompassed by a&gt;, b ', c&gt; or

d). This includes in particular the Eco RI / Sal I insert of L. lactxs of approximately 3.5 Kbp, a fragment thereof encoding the signal peptide MSP and / or the promoter of the MSP gene. Depending on the type of DNA molecule of the invention inserted into the hybrid vector, the vsctor may comprise a hybrid expression control sequence. Such a hybrid expression control sequence consists in part of a DNA molecule encompassed by a), b5 or c&gt; and is partially a DNA molecule different from a DNA molecule encompassed by a), b) or c) = For example, the vector The hybrid may comprise the MSP promoter in combination with a DNA sequence encoding a non-MSP signal peptide, a DNA fragment encoding the MSP signal peptide in combination with a different promoter from MSP. In addition, a hybrid vector of the Invention optionally comprises a transcription terminator region, eg the E. coli trpA transcription terminator. Hybrid vectors comprising a DNA sequence encoding a signal peptide of the present invention, ie derived from the pre-MSP gene or a related gene, are useful in producing gene products secreted in lactic acid bacteria, particularly in tact oc oc uss, especially in L. lactis- or L. cremoris or other bacteria, for example in a Baci11us spec. such as 6 thuringensis. Such a hybrid vector comprises a promoter that functions in the target bacterium, for example the MSP promoter for lactic acid bacterial expression and a culture-transcription termination region that functions in the target bacterium. The use of a terminator may increase the yield of the recombinant gene product if it is operably linked to its homologous structural gene or

<img file="PT97081B_D0024.tif" />

hs Lerologue »By BJiSiiipiGj S6 u t.siflHXnach-Jf 'ti' jjA of IO, COi 1 SstlYSf operably linked to a hirudin tene which is expressed under the control of the MSP promoter and a DMA molecule encoding the signal signal peptide. MSP in lactic acid bacteria? “Desulfate-hirudin production is markedly increased»

A hybrid vector of the invention also comprises an origin of replication that functions in the intended host? and g »lactic acid bacteria and / or Bacillus spec» and / or E «coli»

<img file="PT97081B_D0025.tif" />

A DMA sequence bearing a origin of replication at least in lactic acid bacteria is for example the 17.7 kbp Cia1 fragment from plasmid pSH71 (Gasson and Anderson ™ 1985) or one of the DNA fragments defined herein comprising the origin. 5Md or plasmid 5 = 2 Md of replication of L »IactisLL.712»

A hybrid vector of the present invention may contain a selectable marker of choice. brand depends on the host you want to transform? select and clone »Any marker gene that facilitates selection of transformants may be used due to phenotypic expression of the tag. Suitable markers particularly those expressing resistance to. antibiotics? and »against erythromycin? tetracycline or ampicillin or in the case of auxotrophic mutants? which complement host lesions, and which are the lactose metabolizing pathway, preferred embodiments of the Sb vector, comprising a scrutinic gene, are homologous or heterologous? by way of example the MSP or especially a heterologous structural gene defined herein which is operably linked in the reading sequence

DNA coded ad peptide sins! MSP promoter MSP or from

<img file="PT97081B_D0026.tif" />

and which may be expressed under a heterologous promoter.

Structural genes may be derived from viruses, prokaryotic cells or eukaryotic cells and may be derived from genomic or cDNA DNA prepared by the m.RNA pathway or may be chemically synthesized; they may encode one;

What is the variety of useful polypeptides, including glycosylated polypeptides, in particular from higher eukaryotes, especially mammals, animals, or human esoecialments, such as enzymes which may be used, for example, for the production of and for carrying out useful polypeptide reactions?

hormones, nutrient polypeptides

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<td>important for</td><td>ot</td><td>ratification</td>

<td>s or. from anima</td><td>is,</td><td>for example</td>

<td>properties</td><td>immun &lt;</td><td>omodu1ador</td>

Siiii-virsis eri ... -jumumoraxs, arrocxoroos, viral antigens, vaccines, coagulation factors, foods and the like.

Examples of such heterologous structural structures are eg hormone encoders such as secretin, thymosin, relaxin, caicitonin, luteinizing hormone, parathyroid hormone, adrenocorticotropin, hormones atherosclerinocytes.

β-Ιipotropin, uragsstrons or insulin, growth factors, such as Tscxor a and epidermal growth, insulin-like growth factor (IGF), and „q. IGF-1 and IGF-II, Qlia-derived nerve cell growth factor (CTGF-transforming growth factor), such as TBFp, growth hormones such as bovine or human growth hormones, interleukin such as (or -2, human macrophage migration inhibiting factor (CMIF)) interferences such as human immunostaining, e.g.

Interferon Interferon Qaraa or a hybrid interferon. by 'J

<img file="PT97081B_D0027.tif" />

'-ί-Λ? li &lt;xB ”&lt;xD, speci. Proteinase users, for example, a hybrid interferon 'm' '' ''. * ', I; - ·' 'Vcay ^ cavSf ·· * H ~ hri rlr * i Q: V \! 3T3 á · - * · · «Í - * ~. -.-.-.

mterThere will be na.dr3.ba isuats cq -antitripsin, SLPI-and the like, hepatitis virus antigens, such as hepatitis B virus surface or nucleotide antigen or non-A-not B hepatitis virus antigen, activating DiasffiinoqoniG activators tissue-like plasminogen or urokinase, tumor necrosis factor®. 1, somatostatin, renin, Is-ertdorfins, immunoglobulins such as

D, E or 8₃ or human immunoglobulin binding immunoglobulin, hepatitis A

Ι-5ΙΞ- CAKE

4-,·

<td>heavy</td><td>i mun oq 1 o bu 1 ina</td>

<td>human. — mu-rg</td><td>factors</td>

<td>as an actor</td><td>connecting to</td>

<td>nina, eg pt d</td><td>• and the related</td>

as factor IX or VHIc, erythropoistin, egline such as egline C, hirudin, desulfate-fhirudine, such as HV1, HV2, HV3, ELeu, ire variants^-J — HVl, ELis^rDesulfate 3-HV2 — hirudin or PA, human superoxide dismutase, viral thymidine kinase, β-lactamase, glucose isomerase. A preferred gene is the coding for. dessulraw-niruoina,

t. 4In the hybrid vectors of

In the present invention, a promoter and / or MSP signal peptide coding molecule is operably linked to the polypeptide coding region to ensure effective expression of the polypeptide.₀

Preferred hybrid vectors are pUCRS, M13 mplQRS, M13 mplRH, pVAHIR, pVAHIR-1, pSCisHIR, pSC12HIR-i and in particular pSC12HIRTerm. The hybrid vectors are described below in the Examples. The invention also relates to a DMA molecule which

a) is the tcoRI / Sa.11 insert of approximately 3.0 Kbp of

L, pUCRS plasmid lactis or a functional fragment thereof

<img file="PT97081B_D0028.tif" />

b) hybridizes to said insert or to a functional fragment thereof or comprises a promoter region that is operably linked to such a hybridizing DNA sequence; it is a degenerate sequence a DNA sequence that is

a) or b) and which encodes a signal peptide or

d) is a derivative of a DNA molecule encompassed by

c) and / or

s) comprises the origin of replication of the IC) plasmid 2.5 Md of L lactisLL712 or ds (II) plasmid 5 = 2 Md of L lactisLL.712 or ds (III) a plasmid of the same mismatch group as plasmid actisLL / 12 rtd or to the plasmid 5 = z Ma of L. «1ac per se» These are ssSd DNA molecules useful for hybrid vectors of the invention or for the preparation of those of DNA libraries.

DIM.A or mRFMA relatively to DNAs or similar mKNAs.

Process for the preparation of DNA and MSP molecules

<td>a</td><td>another objective</td><td>of</td><td>invention is</td>

<td>preparation d</td><td>s a molecule of</td><td>DNA</td><td>of the invention</td>

<td>hi r or d</td><td>s a ds molecule</td><td>DNA</td><td>aaui I defined</td>

... ...

giving:

For that of a vector, comprising a culture of a host comprising a DNA molecule of the invention and isolating a DNA molecule of the invention from such a host in culture or

B) Preparation of a DNA molecule of the invention by an in vitro synthesis.

<img file="PT97081B_D0029.tif" />

i '1'

The culture of the host is carried out in a conventional nutrient medium which may be compounds which allow the negative or positive selection of the transformants to be supplemented with or removed, ie such hosts containing the desired DNA moiety together with a selection mark. Among the non-transformants, the non-transformants include the DNA molecule present.

Any suitable transformable hosts useful in the art may be used, for example suitable bacteria such as gram negative, positive bacteria, eg Baci11us.spec »speci ac tooac ι i us and» q »L-1 ac cis, t. xaccis

<td>E. coli.</td><td>or</td><td>bacteria- Sram</td>

<td>or bacteria</td><td>of</td><td>Cq X · ~ = Ο i -SQ t ICO it</td>

<td>f; f? .i. * f C &lt;5</td><td>imen</td><td>i L - 3.CZLOQQQQU.S ·</td>

<td>d .1. ac and t. i 1 actis</td><td>QU</td><td>L «cremoris»</td>

<td>formed by</td><td></td><td>IBÉzt-OÚQ CChHVSn—</td>

As for the transformants, the transformants were identified in a clonal way.

eg »ps ia

- »It is resistance, for example against tetraci

In particular the hybrid vectors described have been propagated in suitable E. coli host strains such as TB1, HB10I._? JM109, KH1 and the like or suitable Bacillus strains or suitable lactic acid bacteria, eg Lactococcus strains such as L. 1actis0230, L. lactis diacis tis, L. cremoris and the like. The hosts were transformed and selected. by conventional methods Propagated plasmid DNA is isolated from bacteria by conventional methods, for example as described by Birnboim &amp; Doly (1979).

A DNA molecule of the invention may also be prepared by in vitro synthesis according to conventional methods. In vitro synthesis is especially applicable for

<img file="PT97081B_D0030.tif" />

<td></td><td>DNA from the MSP qene or a related coding qene</td>

<td></td><td>or in particular the smal peptide »</td>

<td></td><td>A DNA molecule of the invention may be obtained.</td>

<td>O</td><td>of a lactic acid bacterium containing such a molecule</td>

<td></td><td>particular from your genomic library or</td>

dtí prOfiV

Ofc;

ΙδίδΒκίϊΙ mRNfi:

IMA tor ti r

Sffl

If a more detailed description of the preparation is a DNA molecule which is the EcoRI / SalI insert of L,

Approx. CQIO © 3.5 Kbp from plasmid oUCRS qenofnxc

As a starting material it can be used; if Lactococçus spec ', and' q, L.

a liquid which is prepared according to conventional methods, eg. partial digestion of the qenomic DNA of an L. strain. lactis' and 'q' LM 0230, C2 or LL712, with a restriction enzyme, eg Sau3A or Mhol and cloning of high molecular weight DNA fragments into a suitable vector, and 'g' the E. coli plasmid pUN121 or a phage lam hd a, and q. 1amhd to EMBL3 «

<img file="PT97081B_D0031.tif" />

<td></td><td>Any other</td><td>strain of</td><td>acid bacteria</td><td>JLV10</td><td>1 tl! _! J</td>

<td>producer</td><td>MSP</td><td>S5 F * V 3. r Ο ΟίΠΌ</td><td>source for</td><td>bibl</td><td>yoteca</td>

<td>genomics</td><td>and like you or yourself</td><td>rooveotoreo</td><td>suitable power »</td><td>θ (Γ ·</td><td>used</td>

as recipients of the fragments.

Genomic screening for sequences * requires a DNA probe to bifar and, with the structural gene nSP = Est · synthetic if the sequence of e = g »is known» Like neither protein

Successful MSP from the DNA library of the present invention is ideal with such a DNA sequence, c. Tlc *. tc-C? F ~ iGcd CsS?

Qsní? MSP or psrts two for s. sequence of the MSP qene or

P -artsds I were known

<img file="PT97081B_D0032.tif" />

MSP s from the preparation of hybridizing UNA probes were first reported 1 »

MSP is the produc

The ujest.

Isis abundant in the supernatant and lactis as assessed by electrophoresis in which staining of the □ by precipitation with TcA of the supernatant? of SDS — polyacrylamide of precipitated proteins with bright Coomassie blue

For the purification of MSP any source may be used, for example the supernatant from a lactic acid bacterial culture, and Lactococcus soec. such as L. 1ac tis. MSP can be purified from a SDS — polyacrylamide gel by cutting off a piece containing the main protein band from the gel and eluting it »MSP»

<td>LlLl t. ~ Í iJ'cõ</td><td>methods</td><td>of purification 3</td><td>such as</td><td>prec xpi Tie them</td>

<td>acid acid, © s Q «</td><td>acid t</td><td>laughs at chloroacetic,</td><td>tion of</td><td></td>

<td>precipitation in</td><td>form of</td><td>LuTí 3-31 NEXT</td><td>dxá1ise,</td><td>C r * O7'- ~ iOr r ~ 3. '</td>

<td>fia, e = g = chromatography</td><td>Qrsfis</td><td>of affinity, cr</td><td>Giftâ t OQ Γ 6. fi S.</td><td>from per mu t:</td>

ionization, gel permeation chromatography, electrophoresis, eg with SDS · polyacrylamide, isoelectric focusing, electroelution and the like or any cumbiuation applied in order to obtain pure MSP or any other.

In the present invention, an MSP with an apparent moiety of approximately 70 kU was isolated in pure form by trichloroacetic.

Ac idot ric i oroacético pain precipitated protein on a gel region with one major band troforese e1ec .tbsequen thee ds ADS-polyacrylamide cutting protein, e 1 c astrocytomas MSP uindo

V fc 1 -5C X QFifii-5 5

UUlií

<img file="PT97081B_D0033.tif" />

MtiP having another molecular weight - apparent amino acid sequence of MtiP was partially determined by sequencing and partially deduced from DNA sequence. It is shown in the sequence listing in 3EQ ID NS1 and extends from position 28 to 466 of the sequence Amino Acids The pure MSP protein was also the subject of the invention,

Sequencing of the N-terminus of MSP was performed in conventional manner and revealed the following amino acid sequence? X-X-Asn-Ser-Asp-11 ε-A1-a-Lis-S1 n-Asp-A1 a-Tr-11 e-Ser-X-A1 a-G1 π -Ser-Ala ™ Lis-Ala —Gin — Wing — Gin — Wing — Gin — Going — Asp, The two next amino acids and the amino acid at position 15 that are indicated with have not been detected,

Based on the amino acid sequence at position 5 to 13 the following oligonucleotide mixture was synthesized?

GAif-L AlN._. Gt_- 1 OAtM ϊ5ΑΝ_Λ idOx AC_and In this nucleotide sequence

2 -3 -3 1 'of the. is T or Cs N, 'is T, C or As N_. is A or G »A represents a * Λ, -f * *¹ Adsnxna-based nucleotide, T with Timxna, C with Citidine, G with Guanosine and 0 with Inosine. The oligonucleotide mixture was radiolabeled in a conventional manner and used to make Lactococcus spec ', particularly L' lactxsLM © 23 ©.

miss a genomic library

DNA probes containing amino acid sequence coding sequences for MSP and having at least about 14 bp can be used for nucleic acid screening comprising related MSP 'genes or parts thereof, which also include ro.FINE screening. encoder

For screening purposes the DNA probes were radiolabeled at their 5^? by methods known in the art

Using p-Aip gamma and T4 ds kinase. The nucleic acid-bearing host or phage microorganisms of the present invention as an insert were identified by hybridization with the labeled gene probe of the gene library filter replicas.

The hybridization conditions used were conventional and may be more or less stringent, eg simply by choosing different temperatures.

The hybridizing portion of a library cDNA clone that hybridizes to the oligonucleotide mixture has been partially sequenced from sct1rtID with conventional methods. The sequence determined comprises almost the entire MSP functional gene. The sequence is described in sequence listing SEQ ID NQ1. ,

The 3.5 kbp L-lactis EcoRI / SalI insert can be isolated from positive clones of a genomic library by digestion with EcoRIs Sal and subsequent purification of the insert according to conventional methods and can be ligated into a suitable vector, eg »in M13mp18 to generate M13mp18RS or in pUC8 to generate pUCRS.

Likewise, any other L. lactis insert comprising MSP sequences, for example larger derivatives, 3.5 kbp EcoRI / SalI insert variants or fragments or DNA molecules hybridizing to the 3.5 kbp EcoRI / SalI insert or which comprise a promoter region that is naturally linked to such a hybridizing DNA molecule can be isolated from a lactic acid bacterial genomic library.

'-if'

<img file="PT97081B_D0034.tif" />

Fragments of Suordo uo® ajci DNA mulèeulci can be obtained conventionally, eg by isolation of the fragments after digestion of the insert with suitable exo- or endonucleases, e = g = with exonuclease III, Ba.131 or SI or restriction endonucleases such as Sau3A, HindiIX and the like. Fragments may also be obtained by in vitro DNA synthesis according to conventional methods.

The mutants of. DNA molecule encompassed by (a) to (c) herein having inversion, deletion, insertion or point mutations may be prepared according to conventional methods by direct mutagenesis (see review article by Zoller and Smith 1983, Botstein and Sbortle, 1985 or Morris et al., 1983) using mutagenic primer oligonucleotides or by deleting DNA fragments between the two restriction sites by cutting with the appropriate restriction enzymes and reliquing the DNA, optionally in dilute solution »

Following is a detailed description of the preparation of #

A recombinant DNA molecule according to the invention comprising a DNA sequence that functions as an origin of replication (according to conventional methods, e = q = as described by Bs Doly C1979) with the modifications described in the examples and separated from them. in conventional manner, using chromatography techniques, for example Mania chromatography. Tis et al.

/ XXUB irnboim iif cí.fir

ΓΌΠΙ-S to— '

Agarose gel la as described in (1982). Plasmid 2 = 5 Md ε and plasmid 5 = 2 Md were isolated and fragmented according to conventional methods »

- ·. &lt;C λ

Mixtures of the fragments thus obtained were ligated with a suitable vector according to conventional methods, and as described in Maniatis et al. (1982) and these ligation mixtures were used to conventionally transform an intermethyl host strain. -

<img file="PT97081B_D0035.tif" />

A suitable vector carries a genetic tag for selection in lactic acid bacteria, and, a resistance tag gene, for example the erythromycin resistance gene and an origin of replication that does not work in lactic acid bacteria. but rather in a suitable bacterium as an intermediate host for cloning of L. plasmid fragments A suitable vector also comprises a marker gene for selection in the intermediate host which may be identical to the marker gene that functions in the lactic acid bacterium. An intermediate host is for example an E. coli strain, and An appropriate cloning vector is then a coli vector, for example a pUCI derivative carrying an erythromycin resistance gene, such as pUC-383, the construct of which is described in the Examples.

<img file="PT97081B_D0036.tif" />

Intermediate host cells that are transformed with a vector comprising 2.5 Md or 5.2 Md plasmid fragments of L. lactis may optionally be conventionally selected depending on the type of intermediate host cell and the vector used. Selective markers may be for example resistance markers or genes encoding a defectible enzyme, e.g., the product of the EREI lacZ gene, β-galactosity, provided that the host is an E. coli strain. defective in the genomic lacZ gene »Single-tag genes can be disturbed by insertion of a DNA fragment» If the tag is lacZ, host cells carrying a vector with a fragment inserted into the lacZ gene are no longer capable of converting X-Gal to a blue dye and as a consequence, positive clones in X-Gel containing ag-ag plates remain white upon induction of the expression of the gene. lacZ gene with IPTS »

Vectors carrying a 2.5 Md or 5 = 2 Md plasmid insert of L. lactis were tested for their own. ability to replicate in a plasmid-free L. lactis strain, s = g = in L. To this end the plasmids were isolated from an intermediate host and used to transform L = lactis 0230 cells according to conventional methods, and as described by Powell st al, (1988). replication vectors comprise a DNA insert derived from plasmid 5 = 2 Md or 2 = 5 Md of L = lactis that functions as the origin of replication. A DNA molecule comprising this function may be isolated from the replication vectors, fragmented, It is mutagenized and subjected to other similar treatments and used to construct recombinant DNA molecules according to the invention, eg »cloning or expression vectors that replicate in lactic acid bacteria.» Origins of replication derived from plasmid 2 = 5 Md or 5 = 2 Md of L = lactis are also functional in bacteria other than lactic acid, and 'g' sm Bacillus spec 'or E' coli '. vectors comprising such DNA fragments may be used as shuttle vectors.

Plasmid fragments 2 = 5 Md or 5 = 2 Md carrying a replication origin can also be identified and isolated according to the method described above from a fragment mixture that was obtained by fragmentation of the total L plasmids. 1actisLL712 'Cloning fragments carrying a origin of replication derived from the 2.5 Md or 5 = 2 Md plasmid can be determined by hybridization of the cloned fragments to the plasmids of the t-derived plasmid pool. lactis L.L712 which were separated on an agarose gel or by comparison of the

<img file="PT97081B_D0037.tif" />

support him with ο

2.5

Recombinant DNA molecules of the invention which comprise all of. DNA sequence of plasmid Md or 5.2 Md of L. lactis or a plasmid of the same incompatibility group of plasmid 2., 5 Md or 5.2 Md can be obtained for example by cutting the respective plasmid with an endonuclease of suitable restriction and ligating it for example with a DNA fragment comprising a homologous or heterologous structural penis, a promoter region or vectors sequences or with an adapcaoor or similars have only one olasmid site.

Restriction endonucleases t_O i sTicc⁵} i UtJ appropriate ulxvsqeHi in respecc

A sequence molecule with the function of Md-derived replication origin can be obtained for example by a method comprising preparing the L plasmid pool. , from plasmid 2 = 5 Md or 5 = 2 Md, eg by calculating molecular weight by agarose gel electrophoresis, fragmentation of respective plasmid, and = q = with suitable restriction enzymes, preparation of a fragment comprising the origin function diípl «S = G ::

one of the fragments referred to hereinbefore and ligation of such a DNA molecule fragment comprising no origin of replication, a cloning vector without its origin of replication, and selection of DNA molecules that replicate in at least one. lactis and in E. coli.

Plasmids comprising an origin of replication of the same group as 2.5 Md or 5.2 Md incompatibility of plasmid

<img file="PT97081B_D0038.tif" />

could be identified because they could not be kept together with plasmid 2.5 Md or 5 = 2 Md, respectively within the same cell. The invention also relates to the use of a DNA molecule or a recombinant DNA molecule of the invention for the preparation of hybrid vectors for expression of a structural gene Examples of such structural genes are given below. Hybrid vectors can be prepared according to conventional methods using enzymes such as restriction enzymes, DNA polymerases, DNA lyases and the like.

Examples 4 and 5 exemplarily and in more detail describe the preparation of viorior hionoes for the expression of a structural gene in lactic acid bacteria.

particularly in L. lactis or Bacilli meni. thurir.qiensis.

Hosting the three-process processes for their preparation

The invention relates to a bacterial host transformed with a hybrid vector of the invention.

<img file="PT97081B_D0039.tif" />

Q bacterial hosts transformed according to

<td>the invention is to</td><td>suitable for</td><td>3 C</td><td>lonagem,</td><td>a &lt;n^:</td><td>simplification and</td><td>^!/OR</td>

<td>preparation of a</td><td>hybrid vector</td><td>coinpr</td><td>eenoendo</td><td>an</td><td>molecule of</td><td>DNA</td>

<td>rifôfxnids &amp; Π1 â) - £ »</td><td></td><td>ss hx</td><td>pc bridos</td><td>idem</td><td>Γ3P1Í CSΓ “S3</td><td></td>

selective response in the host population that may be contained in the hybrid vector.

an E. coli-typed DNA sequence, Bacillus lustal hospeoeirus and no cell s-lost during proliferation, used depends on origin of replication In case the hybrid vector comprises a ro replication origin function -suitable is for example any es spec = or Lactococcus spec. that does not contain a plasmid with an origin of replication of the same incompatibility group »

Where the hybrid vector comprises a homologous or heterologous structural gene fused to a correct reading frame with the DNA sequence encoding the MSP signal peptide, a transformed host of the invention is such that it is suitable for the production of a homologous or heterologous secreted protein. , and »g» a strain of L = lactis »

An example of a co-transformed host according to the invention is a strain of E. coli, and TG1, C60®, or HBl1 transformed into pUCRS, pSC12, pSC12deltaP, pSC12deltaN, pSC12deitaMP, pSCIS, pSCidELVAH, pSCiDELtaH -1, pSC12HIR, pSC12HIR-1 or pSC12HIRTerm or L. cremoris or L 'lactis without plasmids, eg' L 'lactisLM025 © or a Bacillus strain,

eg »Eh_thurinqíensis transformed with one of these plasmids» E »coli ΊΈί transformed with pECRS» L »lactis transformed with pUCRS, pSC12HIRTerm, pSC12 or pSCÍS and B» thurinqiensis transformed with pvAHIR or pVAHIR-1 are also preferred. for the preparation of such transformants comprising treatment of a host under conditions of transformation with a recombinant DNA molecule of the present invention, especially a hybrid vector of the invention, optionally together with a selective marker gene and transformant selection »

D ^ Cssso._par ^^ _ j3r (^ ration_Ljs_. £ 2112 ^ t.

The invention further relates to a method for the preparation of a polypeptide characterized by a structural gene.

<img file="PT97081B_D0040.tif" />

homologous or heterologous is fused in the correct reading frame with the MSP signal peptide coding sequence UiMfi, that a suitable gram-positive haterian host for example a lactococus host, e = g. L. lactis LM0230 or Bacillus spe comprising such a gene fused and that the polypeptide encoded by said gene is secret. by host cells ·:

When used, the polypeptide is isolated from the supernatant according to conventional methods. In a covert embodiment a protease on its protein is expressed as a protein degradation to the desulfate-hirudin, which are secretaries by L = 1actistM0230 cells transformed with a hybrid vector of the invention comprising a gene encoding such a protein, and either gpSC 1 2HIR, pSC 12HIR-1 or in particulate powder. . m. L. * Oi icen ions LS uessui Tau_? niruaina -áLLivi could be obtained from the supernatant of stationary phase cultures of pSC12HIR or p-SC12HIR-transformed L. lactisLMo25®t cells. The supernatant of pSC12HIRTerm-transformed L. lactis cells increased the concentration. Surprisingly,

Such an achievement may be pious.

protein production.

desulfate-hirudin levels of the heterologous gene products, and 'g' desulfate-hirudin, do not decrease after prolonged incubation, and 'g = overnight of stationary phase cultures indicating absence of heterologous gene products in the supernatant.

In another embodiment of the invention cells may be harvested from the culture medium in the log phase or stationary phase, and 'g' by centrifugation or filtration and resuspended in a smaller volume 'and' g 'in about 1 s. 20Z of

<img file="PT97081B_D0041.tif" />

original culture volume, fresh nutrient medium or suitable buffer solution. Incubation of the resuspended cells results in increased yield of the heterologous gene product that is secreted into the supernatant. For example, in a culture of L, lactis LM®23® transformed with pSC12HIR an increase in the amount of desulfate-nirudin in the supernatant of a culture can be obtained. stationary phase are harvested? resuspended in about 1 / 1® of the

Sj S? -cí tí t? j. tA X ct fzj by centrifugation and incubated for about 60 min. about iUi Uf X i * ·

Another embodiment of the invention is the production of MSP which is secreted by positive host cells transformed with a hybrid vector of the invention, eg pUCRS, according to the methods described above.

Another embodiment of the invention is the production of desulphate-hirudin in B = thurinqiensis, preferably in the pVAHIR or pVAHIR-1 transformed HDI strain: rYB CDSM 4574).

Brief description of the sheets

Figure 1 Physical map of the plasmid publish map.

are approximate and given in Kbp. The pUC1Q fraction extends from the map position ® to 2.7 and the pVA838 fraction of the erythromycin resistance qene extends from the map position 2.7 to 4.4. , The restriction sites shown in parentheses were destroyed during the preparation of the resistance molecule. '' - - -&gt;

cina; recombinant ori. Are the meanings of abbreviations amp ampicillin? ery * erythromycin resistance gene origin of replication derived from oUCIS.

<img file="PT97081B_D0042.tif" />

Figure 2s Physical map of plasmid pSC12. Map positions are approximate and are given in Kpb. The DNA of pUCJS extends from position v to position 2.7. The 1.7 kfodo pvA838 erythromycin resistance-bearing fragment extends from position 2.7 to 4.4. The insertion derived from L. lactis plasmid 2 = 5 Md extends dBSBs to position 4.4 ax 8.0 / v. The restriction sites shown in parentheses were destroyed during the preparation of the recombinant molecules. The meanings of the abbreviations are PL1, region of the EcoRI site pUC polyadaptor to the site CSmal). F'L.2, pUC polyadaptor region from the site (Ima) to the Sphl? amp ', ds ampicillin resistance gene? sry, erythromycin resistance gene? ori = pUC-derived origin of replication? origin of replication derived from the 2.5 Md lactococci plasmid as deduced from the analysis of 13 td X 5 O Figure 5s Physical map of plasmid p8C18. The approximate and are given in Kpb. PUCIS DNA extends from position ® to position 2.7. The 1.7 kb erythromycin resistance-bearing fragment of pVA83Q extends from position to 4.4. 5.2 Md of L. lactis' J extends from position 4.4 to position 5/0 »The restriction sites shown in parentheses were destroyed during the preparation of the molecules. recombinant. Meanings of abbreviations are PL1, pUC polyadaptor region from EcoRI site to CSmal) s F'L2 site_? pUC polyadaptader region from site (BamHI) to site Hindi II § amp¹ , erythromycin resistance gene to ampicillins ery^f, resistance gene in the ori

PUC origin of replication derived from pUCs derived from plasmid 5 replication origin = 2 Md lactococci as deduced from deletion analysis.

<img file="PT97081B_D0043.tif" />

i i^; V λ

«Pre ν3. the tur there dhiTF 'deoxynucleotide triphosphate

High resolution liquid chromatography HPLC

IPTG isopropyl thioga 1 ac topi r-year

Kpb Kilopares ds bases k DK i I od 11 on s

LB Luria Nutrient Medium in Broth (Gibco / BRL)

Md Meq a. of solids sCD23 soluble CD23, i e = 25 K IgE binding factor SLPI Inhibitor of secreted leukoproteins

The following examples serve to illustrate the present invention, however, they should not be construed as a limitation of the invention.

Materials and methods

J

<td>L lactisLr</td><td> 10230</td><td>is a derivative without</td><td>ρ1ssfflideos</td><td>from L.</td>

<td>1ac tis C2 E f sta h iou</td><td>J. D «</td><td>and L. (McKav, 1977).</td><td>She was dep</td><td>osita</td>

<td>at Deutsc he Sao 1 unq</td><td>von</td><td>My kr oorq i seien und</td><td>Z and 11 ku 1 you re ·</td><td>n Cver</td>

iaqui sm on)

L 1actisC2 (NCDU2031) and L 1acfisLL712 which later served as the source of pls.s-mid &lt;

<td>Related nts</td><td>or</td><td>i d's'n 11 c os (</td>

<td>was obtained</td><td>per</td><td>example then</td>

<td>ganisms, UK CiMCDD</td><td> 207,</td><td>1) i; this ώ 11</td>

fius v »Md so« 2 Mo, sound narrow. = Davis et al ·. ,, 19S1) = n

Sammlung von Mikroorganismen und ielIkulturen (see below) ε, uoilTBl teflf! the isnophype

<img file="PT97081B_D0044.tif" />

<img file="PT97081B_D0045.tif" />

- i'O · '' ac-proí junith i 5 hsDb / F⁵ traD3á = ρroAB ₅1acI „1ac ide1taM 1 b ii purchased

Amersham? DXiqonucIeotide — directed in

General Methods:

j rans τorsisation rsuada rl sr * π · ··· rs r \ calcium chloride as described in Maniatis et al.

Transformation of the lactococcus strain was performed by electroporation using a BioRad Gene Pulser system and following its L-lactis LM © 23 ©

Plasmid Preparation

The plasmid uNA of E = coli was resected with the method of Birnboiai and Doly (1979) =: oroo. The same method was used for isolating plasmids from lactococci with the following modifications:

(a) Cultures grown at Sadine, 1975) at 30 ° C, diluted for a further two hours in M1 / ​​G medium (erzaghi

b) Incubation with lysozyme was also performed in the presence of mutanolysin (50 mq / ml) for 10-20 min at 37 ° C.

A pool of plasmids was prepared by the same method and the plasmid fraction was further purified by centrifugation.

<img file="PT97081B_D0046.tif" />

96v) »The three smallest plasmids &lt;1.8 Md, 2.0 Md, 5 = 2 Md? Gasson, 198-3) were enriched in the purified plasmid fraction while the two larger plasmids C9Md and 33 Md 5 Gasson,

198-5) are present in smaller quantities

<img file="PT97081B_D0047.tif" />

<img file="PT97081B_D0048.tif" />

• J pVA838 CATCC 37th © | Mac Rins et al »₃ 1982) is a ρ las-mi de ds Istococci carrying the erythromycin resistance gens constitutively expressed in a restriction fragment Av-al / Hind 111 ds 1.7 Kpfa »About ds 1 © μα of pVA838 were digested with Aval and HindIII. The ends of the DNA fragments were blunted with the Klenow enzyme in the presence of four dNTPs. The fragments were separated on an agar gel and the Aval / Hind III fragment_?7Kbp was recovered from the gel by electroelution »

About 2 µg of said Smal-cut pUcltI fragment and µg (Norrander et al.) Were ligated in the presence of T4 ligase as suggested by Rusche et al. and the ligation mixture was used to transform E. coli TQ1. White colonies of LB agar boxes containing X-Salt, IPTS and 1 µg / ml ampicillin were picked. The correct clones were identified by isolation, analysis of their plasmid DNA and growth capacity on LB agar plates containing erythromycin (&lt;1 mg / ml). The resulting plasmid was designated pUC838 = The restriction sites and pUC83S isotopes are shown in Figure 1.

í.2 pSd construction

LUtij Lu ê ut de pxasmíd from L. »lactisLL7í2 am or separated on a preparative gel.

<img file="PT97081B_D0049.tif" />

agarose »The second strand of DNA forms ccc of plasmid 2, = 5 Fiti» It was removed from the gel and was electroeluted. »Digestion of this plasmid with SphI and aqarose electrophoresis revealed a single 3.5 kbp fragment indicating the

<td>iVaUteí »!</td><td>by Sphl</td>

<td>0 Sohl</td><td>and treated</td>

<td>5fc? no</td><td>of vector</td>

<td></td><td></td>

<td>til ds</td><td>3.5 Kbp from</td>

pUCS38 was cut at its single site with calf intestine phosphatase. About phosphatase-treated and 15 µg fragment S plasmid 2.5 Md was ligated with ligase. T ligation was used to transform competent coliTSl cells. 37 * C in 1 LB for 90 minutes and then transferred to 200 ml of

AND"_

IT * t

Jit ~

LB contains 1®® mg / 1 night »0 DMA erythromycin ds: terminates at 3 /^!during plasmid was prepared from cells of this culture and about 2 μα were used to transform lactis LM0230. Transformed L = 1 ac tisLI-1o23o cells were selected.

and Ml / S agar tromycin containing 5 mo / ml of

SJ plasmid DNA fo:

clones of the transformed Iac11sLM0230 cells and subjected to restriction enzyme analysis. All transformants investigated contained an 8.0 kbp plasmid, pSC12, from which the 3.5 kbp SphI insert was recovered. A physical map of pSC12 is shown »pSCi2 can pass coli several times and vice versa without changes in the restriction pattern»

<img file="PT97081B_D0050.tif" />

1.3 Construction of pSClí

DMA of the set jfliídeos dt

Iacxxsi id i / 12 was partially digested with 5au3AZ by incubating amounts of 500 ng of DNA with different amounts of restriction enzyme. Samples containing partially cut DNA were identified on an aggrose gel. This DNA was ligated to the pUC838 vector which was previously cut at its unique site of calf intestine alkaline phosphatase crammed with fc., Coli, thymidine isolation and subsequent transformation of L, lactis LM023® was performed as described for Again, restriction digests were performed on plasmid DNA isolated from various L = lactis transformant clones. They carry an 8.8 kbp plasmid, pSclS »Passing the plasmid several times from L» lactis to E. coli and vice versa did not result in any changes in the restriction pattern indicating construct stability in both cell types. »Fig. 3 shows a physical map of pSC18 »lentiication of lactococuss replication origins by dejection of pSCi2 and the obese

The regions in plasmids pSC12 and p8C18 carrying the origin of replication of lactococci were defined by deletion analysis as follows.

DNA between the appropriate restriction sites was by plating the plasmids with the corresponding enzyme from the restriction enzyme cleavage sites into pSCl2 and pSCIS and recirculated with 14 Ixqase,

The approximate λulcii xzaçcio are shown in Figure Table 1 »

Μ

-J

<img file="PT97081B_D0051.tif" />

's pec cx vamen re?

pBCISdsXt- = Ah^! &amp; p-SClBctelter ΐον'β.ι'π constriddοϊξ- by DNA ligation between the two FvuII restriction sites in the pUC18 part of plasmids pSC12 and pSCIB, respectively »thus removing E. coli derived origins of replication»

In pbC12deltaN and psClódeltaN the smallest fragment

PSC12 mdel of pSul2 »puu jpecci vsffiSs ί ί_ΐ2 5 Torsi» removed EcotIV fragment in

In pSC12tíeltaNH DMA was removed between the F'vulí site in

JC is the Ndel site in the lactococci part of pSC12. In this case the vector DNA was blunt-ended with the enzyme Kleno ^ f &lt;strains of the reaction ».

Another pSCiS derivative? pbL-lSdel taH, has no DNA between HindiΙΣ sites on plasmid »

Mutagenized plasmid DNAs - were first of the E1 coliTG1 transformants that are resistant to erythromycin. It was then tested if they could be used to transform 1,1'actis-Ls402-5.^;3 relatively -a. erythromycin resistance »Absence of transformation was assumed to indicate that Isctococus origin of replication was removed by deletion» When transformants are obtained

IGdO5 OS isolated from or. destroyed erythromycin resistant piasiMeo svamnete xsoiaoo and its structural integrity confirmed by analysis and restriction

For both plasmid

<img file="PT97081B_D0052.tif" />

As a necessary region for replication is identified as paired. From the fact that the TeltaP derivatives can replicate in E. It is obvious that the cloned lactococcal origins can also be found in the gram negative bacterium E. coli.

The results of this experiment are apt.

Table 1 »

Table is Results of anâlií

Plasmid Deleted Region;

Deleted fragment '' po'L i 2 nsnnum pSC12dsItaP pSCÍ2deltaN p&gt; SC 12d and 11 aR

PvuII (0.1), PvuII (2.5) Ndel (0.2), Ndel (6.2)

Ec oR VC 5.0), Ec oRV (7.55 pSC 12 of 1 t-3'ΝΡ Nde I (6.2), Pvu II (2.5) pSC18 no pSClSdeltaP pSC1bde1taN pSCÍsdeltaH

PvuII (0.1), PvuII (2.5) Ndel (0.2), Ndel C7.5)

HindIII O), HindIII (δ, 5&gt;

: Md was lOCDCDS, ml -ia

Tuncioíentaaos

Keplication: plssmíds rtli L

ND 'o em

Iscti

Deletions extend between the two restriction sites. The approximate positions of the restriction sites in pSC12 or pSC18 are shown in parentheses as distances to HindIII in the fraction of p! JC83-derived plasmids which is defined as position (0). They are related to the base positions given in Figure 2. and 3, respectively »

Approximate length of the deleted fragment in kbp.

Not determined »χ τ n

j.

— »4~?

on -the origins of re

<img file="PT97081B_D0053.tif" />

I Λ. UJLULUb

The origin of replication in pSC12 is derived from the 2.5 Md plasmid of L lactis-LL712 as described in the DNA fragment isolation protocol carrying the restriction enzyme cleavage pattern indicates that the origin of replication in ρ-12. SClB derived from plasmid 5 = 2 Md »

"Repeating Pd-12 and PSt-18 in other major lactococci are successfully transformed using both plasmids." Plasmids remain stably associated with the cell population under selective pressure.

3 »Cloning of the gene secreting the main secretion product

Lactis LMÔ233 'solamenfo of the major secreted protein (MSP5 of L. lactis in

... Λ.

1.5 l of a culture grown during lactisLM '3230' grown at 33 ° C in M-17G was centrifuged on a Sorvall GS'3 rotor at 7000 rpm for 23 minutes at 4 ° C.^&amp;The supernatant was harvested and the proteins precipitated by the addition of an equal volume of trichloroacetic acid at 107 ° C and incubated for 30 minutes at room temperature. The precipitate was collected by centrifugation on a Sorvall GS-3 rotor at 3000 rpm at 4 ° C. for 30 minutes. Protein pellets were drained and redissolved&gt; ml SDS sample buffer (aemmli, 1970 /. The sample was neutralized by adding a small amount of 4IM NaOH and then dialysing for 4 hours against 25 1M 4 L

<img file="PT97081B_D0054.tif" />

Tris — HCl dH 6.3, 0.02% 8D3 »The recovered sample had a volume of approximately 4 ml.

- OÍÍÍ The sample to increase volume to 6 mi.

w • J

Proteins were separated by migrating 2 ml amounts of 8% preparative gels of SDS — polyacrylamide (Laemmli, 1970) using a BioRad Protean cell. Small strips were cut along the gel, stained with Coomassie Brilliant Blue and bleached in an aqueous solution containing 1Θ7 methanol and 10% acetic acid. They served as markers to identify and cut the main protein band with an apparent molecular weight of approximately 56 KD which was removed from the gel. MSP protein was recovered from the gel po

----.?----? ------- -------- u ££ - - ³------ 'mM and

150 V for 2 hours using a system containing 2 mM ammonium acetate and 0_s© dialyzed for 43 hours against two seedlings of acetic dialyzed protein migrated in a

2 @ mM, 0.005% SDS »A sample of the

Siotrap and a cap

Biotrap

SDS »Ammonium gelate was 8% SDS-PAGt gel and a single protein band with a very good molecular weight was observed. dexxd KD »odd number of apr from approximately

5.2 MSP amino-terminal sequence analysis

The amino-terminal sequencing of the isolated MSP protein was performed according to conventional methods using a gas phase sequencer (Applied Biosystems Inc. Model 470A) with HPLC quantification of phenylthiohydantoin derivatives of cleaved amino acid residues,

The 2 th amino acid sequence in length obtained is

<img file="PT97081B_D0055.tif" />

AT

-Lis-Ala-Sln-Ala-Bln-Ala-G? Ln-Go-Asp »The first two fluids and the excitement in the pusition 10 are the same indications.

3.3 Synthesis of a mixed nucleonide probe

Based on the sequence of

i .: t UC ··.

<td> ·.£}</td><td></td>

<td>jX · * - X i- ^ ** 7 '</td><td>”A x · Ξν</td>

<td>gone in</td><td>nn · j— ~</td>

<td>4 · fsf € --- ZZ.CO</td><td>£ = .- i</td>

<td></td><td></td>

<td>of a</td><td>zJ X</td>

<td></td><td></td>

<td>. in the 2nd</td><td>ΠίΕΓΟ</td>

<td>. of the 4</td><td>dMTP</td>

<td>^:of</td><td>naked</td>

DX 10

-Sln-Asp-Ala-Tre-Ile, which corresponds to amirx 5 to 13 in the N-terminal sequence of MSP, was projected to be broken down. A mixed oligonucleotide was designed for the detection of a rare laugh. - «=« »r ~_s zc- riss «t rhiae? -er -&gt; - = z τ r ~ #

<td>taken from</td><td>mix of</td><td>ol igc</td><td>jfhXu xtóo ⁵~</td>

<td>SAN. SCI X</td><td>B.C. N₁ it's T</td><td>or C ?,</td><td>K^! · * · ~ R ^ «.__t fcs 1</td>

<td>sit one</td><td>nucleotide</td><td>with</td><td>the base</td>

<td>c itidine</td><td colspan="3">«There CUUS (ÁLító-SnitxS-XHcã SI CQ?</td>

Λ-JC, or A 5 N._T

TN », tJU AAhU CAN-y

Zs Zt and A or G = A represents tiiBxna, C with

3 „4 Construction of a genomic library of

Iactis LM®230

L&gt; N'r

<td>• Sonic was</td><td>JL ~ '1 -S do</td><td>from</td><td>L.</td><td>lactis</td>

<td>.1.do?</td><td>protocol</td><td>used for</td><td>isol</td><td>· 5'Τΐ'Ξ^:Π to</td>

<td>. treatment</td><td>w om 1 i so</td><td>up and mutarx</td><td>□ 1 isi</td><td>Π% · &amp; &amp; E *</td>

<td>during</td><td>two hours</td><td>sa tfâ '-' C with</td><td>prot</td><td>einase</td>

of plasmids at 1 ° C and 1 ° C were 1 µM Tris-HCl pH S, 2D-mM EDTA, 0.05% SDS. DNA was extracted once with phenol / chloroform (1 vols Ivol) and then purified by CsCl density gradient centrifugation.

□ Chromosomal DNA was partially digested with SauOAI and fractionated according to its size in gradient gradients.

<img file="PT97081B_D0056.tif" />

sucrose as described in Maniatis. Gs fragments that were about 1 -2-2 Kpb were collected.

Lambda DNA EMBL3 was cleaved with BamHI and EcoRI and purified by phenol extraction and ethanol precipitation. It was then ligated with the β -2 Kpb fragments in the presence of hexamenacobal toC ϊ 11) chloride to favor a. formation of concamers »

Írusche J, R, et al., 19855. The ligation mixture was encapsulated in vitro using the Stratagene Sigapack Plus system.

<img file="PT97081B_D0057.tif" />

Recombinants were selected by sowing from the library in E. coliG359 CKahn J et al., 19305. A total of about 6 recombinant phages were obtained.

3ft5J&gt; esaiste__Ja_ &amp; jMliateça __ ^ _ggulences ^ of ___ DNA

MSPdelayer.de

<img file="PT97081B_D0058.tif" />

About © © piscas ds lambda. Recombinant EMBL3 were seeded by petri dish (diameter) and transferred to Plaque Screen Membranes (CNfc.N.5). The filters were screened by hybridization to the mixed oligonucleotide probe described in Example 3. 3 which was PjATP-labeled label using T4 kinase according to standard procedures described in Maniatis et al '&lt;19825 =

Positive plaques were identified and subjected to a second round of screening at low plaque density.

DNA was prepared from positive phages, digested with restriction enzymes and subjected to Southern analysis as described in naniatis. Southern blots were screened with oligonucleotide mixture. It hybridized to an EcoRIZSalI fragment of 3.5. Kbp at insertion of L »lactis from a phage

<img file="PT97081B_D0059.tif" />

Positive Further screening assays showed a 2.1 kbp HindIII fragment within the 3.5 kbp EcoRI / SalI fragment which hybridized to the oligonucleotide mixture.

The approximate distances of the different EcoRI cut-off restriction sites from the EcoRI / Sal I fragment of

3.5 kbp were determined by agarose gel electrophoresis after digestion of the fragment with the respective enzymes from appropriate enzyme mixtures. The EcoRI-cut end is about 0.5 kbp from the Hindi II site._s about 2.6 kbp from a second Hindi II site and about 3.15 kbp from a third HindIII site »

3.6 Construction of oUCks

DNA from a positive phage was digested with EcoRI and SalI. DNA fragments were seared at a 0.6% aaarose level and the 3.5 Kbp EcoRI / Sal I fragment was cut from the gel and isolated by electroelution. Three-fold molar excess of this fragment was ligated into pUC19 which was cut with EcoRI and SalI with alkaline phosphatase.

The ligation mixture was used to transient E. coli TSI and transformants were selected on plates containing 100-mg / ml ampicillin. D Plasmid DNA was prepared from the transformants and restriction analyzes were performed for the correct construct designated. pUCRS »

And, colifSl transformed with oUCRS was deposited with Deutsche Sammlung von Mikroorganismen und Zel 1 kuituren ÍES?¹!) =

<img file="PT97081B_D0060.tif" />

j ~ 'r udufct) d © Saur' tion

3.7 Pri η o ip gene sequencing

Sequencing was done by the chaosis termination method CSanqer (c) using USBC Sequensse.

The 3.5 kbp EcoRI / SalI fragment prepared as described in Example 3.6 was subcloned into Mi3mp18. The resulting plasmid was designated as M13mp18RS. The 2.1 kbp HindIII fragment of the 3.5 kbp EcoRIZSa.II 'fragment was cloned into M13mp19, the resulting plasmid is M13mp19Hr. A series of one-way deletions from the Sall extreme end of the M13mpIRS insertion were served by DNA digestion with exanuclease III and SI CHenikoff, 1934, Yanisch-Perron et al.₅ 1985) = Deletions extend about 2 Kbp for insertion. Sequencing of these deletions from the universal primer mplS gave information about the. sequence of a chain. Based on this sequence, a series of synthetic oligonucleotides were synthesized as primers of the second strand sequence. Additional sequence information was obtained by sequencing the exoIII-SI deletions generated from M3mpi9H with the same method. of a 192 bp fragment of the 3.5 kbp EcoRI / SalX fragment is described in the sequence listing sm SEQ ID nS1. It comprises part of the MSP promoter region, the DNA sequence encoding the MSP signal peptide and the mature MSP structural gene.

Sequence analysis of UNA con? about 2kbp in length revealed the presence of a single open protein coding (ORF) grid having 461 amino acid residues. Comparison of the deduced amino acid sequence with data obtained from isolated MSP N-terminal sequencing identified one way mature of. 434 amino acid protein * CLt r ~ -3 acids which is preceded by a peptide formed by?

Λ In the same, grid by 2 / amino acids, the last amino acids have the structure kJi

<img file="PT97081B_D0061.tif" />

pica of a signal peptide. Charged residues were found at the N-terminus while the rest of the sequence consists mainly of hydrophobic amino acid residues. The body in biosuj in the young around the site of cleavage seciue rules derived from other signal peptides CVon Heijne₅ 19835»

4 «Production of recombinant desulphate — hirudin lactis in Lactococcus

4.1 Construction of Desulfate Secretion Plasffiides — Hirudin

Plasmid R13 mplVH contains an Hxndlll fragment of

2.1 Kbp encoding the N-terminal half of MSP including the signal peptide and about 1.5 Rbp of upstream DNA. This plasmid is cleaved at its only Seal 129 bp site downstream from the extreme

COUH of the signal peptide. A fragment

(Ceses encoding desulfate-hirudint genetic information was isolated from plasmid pML3O (published in European Patent EP-A-168 342) and inserted at M13 site mplPH »A fusion in the same sequence DNA encoding the MPS signal peptide and the desulfate-hirudin structural gene was created by removing excess DNA by separating the last amino acid codon from the signal peptide (Ala) and the first amino acid codon of the fact-hirudin (Val) . This was accomplished by genesis with oligonucleotides-in-vitro using at least 11 v 5 H m 8 (2 a. 1 ⁵Amersha-Directed Pd'SbX The 2v bp oligonucleotide comprises the last 14 bp of the signal peptide and the first 15 bp of the hirudin gene. The resulting fusion sequence is described in the sequence listing in SEQ 1D N22, fusion-bearing HindIII fragment was removed.

There is

<img file="PT97081B_D0062.tif" />

Insertion were recovered »The resulting plasmids were designated pSC12HIR and d-5C12HIR ~ 1 respectively»

Another plasmid (pSCÍSHIRTerm) was constructed by inserting the trpA tE E. coli transcription terminator (Pharmacia) into the single HSC site of pSCISHIR about 125 ph downstream of the desulfate-hirudin gene.

55ui ra co-ηχr

During the desulfate-hirudin fusion plasmids were aimed at transforming L. actis-NO5050. Transformants were cultured in M-1.76 medium supplemented with 2/1 glucose in the presence of erythromycin (5 mg / ml) at 30Â ° C.

AND-. n t. - ___ ç. HIJ, TUF CAHI USP; - Culture supernatants were then cultured overnight before taking a second sample, centrifuging, removing the supernatant and freezing as well. Desulfate-hirudxna production was determined with a bioassay »biosay measures thrombin inhibition activity

<td>ask you to — nx r</td><td>udina us</td><td>3-0 b F * Of '? 3 of 3</td><td>harvested · »Following</td><td>'33 sound</td>

<td>presentation ds one</td><td>protocol</td><td>dtE t -— x t-all</td><td>of the biomass which</td><td>it is a</td>

<td>inhibition test</td><td>from thrombin</td><td></td><td></td><td></td>

samples and reagents is ®, 2n Tris — HCl, pH 7. 5 containing t, 0 M NaCl and 0.01 / 1 bovine serum albumin »Thrombin is from human plasma (Protogen AG, Laufelfingen, NS 80-13 —1102), the chlorogenic substrate of thrombin and Lnromosvme To iheehrxnger »riannhexm, product N2 20084-9)» P-nitro-aniline released from

Chromosym TH was measured with a Dynatech dog CNunc, olac microolac reader

<img file="PT97081B_D0063.tif" />

were performed on microtiter microwell plates). Each cavity received; 50 μΐ fí = f 5 = ~: Π Hfi

L 4 ~ ί · carnation, 50 μΐ ds supernatant of unknown concentration with thrombin inhibitory activity, i. .and. dessuitato^-hiruflirsa e gl of thrombin solution »The reaction began by adding 150 μΐ of substrate solution (330 u / ml 5 and the plates were incubated to give an A -„ nm == 0, S ± 0 « 2 in a control cavity.

Both substrate solution and thrombin solution were kept used. A standard curve with known concentrations rLTvr^G’^_'3dess.ulfate ~ hirudin (40. 20. 10, 5, 2 used to convert this to the same actin ..

Qfcf U.FF-S and 1.25 ng / rnl) were OD measurements at concentrations of

No super ad an te

transformed L. lactis LM0230 cells

The same level of desulfate-hirudik activity was determined at the same time. “Hί -.- ·.-Ί .-—. ;:. t.-. sr 1 na. However, a culture in the

i.-4

T-3 '= staphylaria • was increased by some 50% desulfate-hirudin activity level. Supernatants from pSC12-transformed cells served as a control without desulfate-hirudin activity.

Levels of desulphaluhirudin in the supernatant did not decrease after prolonged incubation for 16 hours thereafter at stationary phase cultures. These results indicate the absence of proteolytic protein in sosrenaoante: tis

LMO230.

<td>NtUuâ G-tpGu</td><td>science,</td><td>at</td><td>cells</td><td>were first harvested</td>

<td>ρο rc Sn tr xf xtg</td><td>Ό OQ X £</td><td>rsss</td><td>pantries</td><td>at 1/1 * 0 of volume ds medium</td>

<td></td><td></td><td></td><td></td><td></td>

<td>and χΓίθυ.ο- ”ί.ς: 3ζϋ</td><td>at 3 € s ° C</td><td>dur</td><td>ante 30</td><td>min for a period of</td>

expression = Q bioassay measured a

<img file="PT97081B_D0064.tif" />

V about six times higher level of desulfate-hirudin activity than in the experiments described above. In a more sophisticated form, cell concentration for a period of expression is one way to increase the concentration of secreted product in the supernatant.

5 »Production of recombinant desulfate-hirudin in Bacillus t hu rinqi en sis

5,: 1 Construction of Plasmid pVAHIR Plasmid pVA838 (Macrina st al., 1982) was digested with HindIII and the ds 5, ® Kpb fragment carrying the erythromycin resistance gene and the Qram positive replication source was primed for pamr. 9 * e 'by troeluição bait "□ HindIII fragment containing the desulfatohirudin gene was isolated similarly from the oSCÍ2HIR'

The two fragments were ligated in the presence of T4 ligase and the ligation mixture was used directly to transform L, lactis LM023® by electroporation. Erythromycin resistant transformants were selected and plasmid DNA prepared from them.

Restriction analyzes of these plasmids were performed to identify the correct constructs. Both orientations of the desulato-hirudin Hindi II expression cassette can be used for the production of Desulfophoram obtained.

-hirudin in Bacillus thurinqiensis »Plasmids have been designated as pVAHIR ε pVAHIR — 1 =

<img file="PT97081B_D0065.tif" />

definition of desuliatu — hirudxna oror B thurinqxensis

B »thurinqiensis stretch © HDi crv (DSM 4o74&gt; was xans

PVAHlH-formed using Leutruption W = Schurter et al 1989

<td></td><td>The transformants were</td><td>selected at =</td><td>olacas</td><td>LB</td>

<td>containing;</td><td>20 pg / ffll eritr-omicins</td><td>at 27 ° C or a. 30'-C »</td><td>0 DMA</td><td>ds</td>

<td>plasmid</td><td>τοχ ρ re p ríxa r r 13. r</td><td>from transformantss</td><td>individu</td><td>such</td>

by the same method used for L. lactis. PVAHIR Restriction Analyzes »

The pVAHIR or pVA838-bearing transformants as a control were cultured in LB medium containing 2 µg / ml sritromycin at 27 ° C overnight. The cultures were diluted at 200 ° C to fresh medium and grown at 30 '.^:Cultures were centrifuged and supernatants were tested for desulfate-hirudin activity 7 hours after dilution.

Desulfate-hirudin was detected in pVAHIR-transformed cells, while none in pVA838 control »

Deposited Microorganisms

5D? ccyl-ciO-Sn Cii o measure

Have the following microorganisms been deposited? according to the Budapest Treaty on. Deutsche Sammlung Mikroorganismen und Zellkulturen (DSM), Ma-scheroder Weg D — 3300 Braunschweigs

Hc

DepósiLu Nl coli K12TS1ZpUuRS

DSM 5803

Fsvereiro,

<img file="PT97081B_D0066.tif" />

<img file="PT97081B_D0067.tif" />

Bates, EEM et al. (1989) Applied and Environmental Microbiology 55,2095.

Bemton, WD, and Davis, RW (1977), Science 196, 180.

Birnboim, HC and Doly, J. (1979) Nucleic Acids Res., 7,1133.

Botstein, D. and Shortle, D. (1985) Science 229,1193.

Chassy, ​​BM (1987) FEMS Microbiol. Rev. 46, 297.

Clewell, D. and Helinski, DR (1969) Proc. Natl. Acad. Know. USA, 62.1159.

Davies, FL, Underwood, HM and Gasson, JM (1981) J. of Applied Bacteriology, 51, 325.

De Vos, WM (1987) FEMS Microbiol. Rev. 46,281.

Efstathiou, JD and McKay, LL (1977) J. Bacteriol., 130,257.

Gasson, JM (1983) J. Bacteriol., 154.1.

Gasson, JM and Anderson PH (1985) FEMS Microbiol. Lett., 30,193.

Henikoff, S. (1984) Gene, 28, 351.

Jos, M. (1985) Applied and Environmental Microbiology 50, 540.

Kam, J., Brenner, S., Barnett, L., and Cesareni, G. (1980) Proc. Natl. Acad. Know. USA, 77, 5172.

Laemmli, UK (1970) Nature, 227, 680.

Macrina, FL et al., In Genetic Engineering of Microorganisms for Chemicals, Plenum Press, New York, (1982).

Maniatis, T., Fritsch, EF and Sambrook, J. (1982) Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory, New York.

Norrander, J. et al. (1983) Gene, 26,101.

Norris, K. et al. (1983) Nucl. Acids Res. 11.5103.

Powell et al. (1988) Applied and Environmental Microbiology 54.6.

Oto, R. et al. (1982) Applied and Environmental Microbiology 43,1272.

Rusche, JR and Howards-Flanders, P. (1985) Nucleic Acids Res., 13,1997.

Sanger, F. Milken, S., Coulson, AR (1977) Proc. Natl. Acad. Know. USA, 74.5463. Schurter, W., Geiser, M. and Mathe, D. (1989) Mol. Gen. Genet. 218,177.

Terzaghi, BK and Sadine, NE (1975) Applied Microbiology, 29, 807.

Von Heijne, G. (1983) Eur. J. Biochem., 133,17.

Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene, 33, 103.

Zoller, MJ and Smith, M. (1983) Methods Enzymol. 100, 468.

String Listing

<img file="PT97081B_D0068.tif" />

SEQ ID NO: 21

TYPE OF SEQUENCE? nucleotides with corresponding protein COMF'KlMt.Ml 0 FROM CADLlAs 19z ^ ® base pairs TYPE OF CHAINS double

TOPOLGtí1A 5 Iinear

SOURCE ORGANISM DRISIMALs Lactococcus lactis LM023 © ÍDSri IMMEDIATE EXPERIMENTAL SOURCE? PUCRS Plasmid (DSM 5803)

FOSIyíLS ND SENOMA; CHARACTERISTICS?

: romos £ 410 part of promoter region 411 to 491 signal peptide MSP

179-5 p ro ts ί at nsadur 'OSSsyO}

PROPERTIES Gene of the main product 1aceis = &lt;Rh ”ó)

<img file="PT97081B_D0069.tif" />

TTTAGGTATT TACGGAATTG CGACCTTATT

ATTGCTCTTT TTGTATATAA TATACAAATA

CTAATCGCTG GACAAGGCTT TTTACAACAA

GACCGCTTTT GAAGTTTTTA GTGCAATCAT

TTTGGATTTG CCCAACTTCA GTTTATCAAA

ACCAGTTAGC GCCTACACTT TTGCTCAATA

TGTAGCCTTA CAATTCCCTT TAGAAATCTT

AAGAAAAGTC ATGTAAGATA CAATTAGAAA

<td>GTTCCCACTT</td><td> 40</td>

<td>ACTATATTTA</td><td> 80</td>

<td>TTATTATTGT</td><td> 120</td>

<td>TATGACAGCT</td><td> 160</td>

<td>TTTGTTGTTT</td><td> 200</td>

<td>TTATCTTAGC</td><td> 240</td>

<td>TTACAGATTA</td><td> 280</td>

<td>GTGTTTTGTA</td><td> 320</td>

'O

<img file="PT97081B_D0070.tif" />

<img file="PT97081B_D0071.tif" />

<img file="PT97081B_D0072.tif" />

<img file="PT97081B_D0073.tif" />

ATCATAAAGA AATATTAAGG TGGGGTAGGA ATAGTATAAT 360

ATGTTTATTC AACCGAACTT AATGGGAGGA ΑΑΑΆΤΤΑΑΑΑ 400

AAGAACAGTT ATG AAA AAA AAG ATT ATC TCA GCT Met Lys Lys Ile Ile Ser Wing

434

<td>ATT</td><td>TTA</td><td>ATG</td><td>TCT</td><td>A CA</td><td>GTG</td><td>ATA</td><td>CTT</td><td>TCT</td><td>Gct</td><td>GCA</td>

<td>Ile</td><td>Read</td><td>Met</td><td>To be</td><td>Thr</td><td>Go</td><td>ile</td><td>Read</td><td>To be</td><td>Allah</td><td>Allah</td>

<td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td><td></td><td></td><td></td>

<td>Gcc</td><td>CCG</td><td>TTG</td><td>TCA</td><td>GGT</td><td>GTT</td><td>TAC</td><td>Gct</td><td>GAC</td><td>A CA</td><td>AAC</td>

<td>Allah</td><td>Pro</td><td>Read</td><td>To be</td><td>Gly</td><td>Go</td><td>Tyr</td><td>Allah</td><td>Asp</td><td>Thr</td><td>Asn</td>

<td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td>

<td>TCA</td><td>GAT</td><td>ATT</td><td>Gct</td><td>AAA</td><td>CAA</td><td>GAT</td><td>GCG</td><td>A CA</td><td>ATT</td><td>TCA</td>

<td>To be</td><td>Asp</td><td>Ile</td><td>Allah</td><td>Lys</td><td>Gin</td><td>Asp</td><td>Allah</td><td>Thr</td><td>Ile</td><td>To be</td>

<td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td>

<td>AGC</td><td>GCG</td><td>CAA</td><td>TCT</td><td>Gct</td><td>AAA</td><td>GCA</td><td>CAA</td><td>GCA</td><td>CAA</td><td>GCA</td>

<td>To be</td><td>Allah</td><td>Gin</td><td>To be</td><td>Allah</td><td>Lys</td><td>Allah</td><td>Gin</td><td>Allah</td><td>Gin</td><td>Allah</td>

<td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td>

<td>CAA</td><td>GTT</td><td>GAT</td><td>AGC</td><td>TTG</td><td>CAA</td><td>TCA</td><td>AAA</td><td>GTT</td><td>GAC</td><td>AGC</td>

<td>Gin</td><td>Go</td><td>Asp</td><td>To be</td><td>Read</td><td>Gin</td><td>To be</td><td>Lys</td><td>Go</td><td>Asp</td><td>To be</td>

<td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td>

<td>TTA</td><td>CAA</td><td>CAA</td><td>THE AG</td><td>CAA</td><td>A CA</td><td>AGT</td><td>ACT</td><td>AAA</td><td>GCA</td><td>CAA</td>

<td>Read</td><td>Gin</td><td>Gin</td><td>Lys</td><td>Gin</td><td>Thr</td><td>To be</td><td>Thr</td><td>Lys</td><td>Allah</td><td>Gin</td>

<td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td>

<td>ATC</td><td>Gct</td><td>AAA</td><td>ATC</td><td>GAA</td><td>AGC</td><td>GAA</td><td>CGT</td><td>AAA</td><td>GCA</td><td>CTT</td>

<td>Ile</td><td>Allah</td><td>Lys</td><td>Ile</td><td>Glu</td><td>To be</td><td>Glu</td><td>Arg</td><td>Lys</td><td>Allah</td><td>Read</td>

<td> 75</td><td></td><td></td><td></td><td></td><td> 80</td><td></td><td></td><td></td><td></td><td> 85</td>

<td>AAT</td><td>Gct</td><td>CAA</td><td>ATT</td><td>Gct</td><td>ACT</td><td>TTG</td><td>AAC</td><td>GAA</td><td>AGT</td><td>ATC</td>

<td>Asn</td><td>Allah</td><td>Gin</td><td>Ile</td><td>Allah</td><td>Thr</td><td>Read</td><td>Asn</td><td>Glu</td><td>To be</td><td>Ile</td>

<td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 100</td><td></td>

<td>AAA</td><td>GAA</td><td>CGT</td><td>A CA</td><td>THE AG</td><td>A CA</td><td>TTG</td><td>GAA</td><td>Gct</td><td>CAA</td><td>GCA</td>

<td>Lys</td><td>Glu</td><td>Arg</td><td>Thr</td><td>Lys</td><td>Thr</td><td>Read</td><td>Glu</td><td>Allah</td><td>Gin</td><td>Allah</td>

105

110

467

500

533

566

599

632

665

698

731

764

<img file="PT97081B_D0074.tif" />

<img file="PT97081B_D0075.tif" />

<td>CGT</td><td>AGT</td><td>Gct</td><td>CAA</td><td>GTT</td><td>AAC</td><td>AGC</td><td>TCA</td><td>GCA</td><td>A CA</td><td>AAT</td>

<td>Arg</td><td>To be</td><td>Allah</td><td>Gin</td><td>Go</td><td>Asn</td><td>To be</td><td>To be</td><td>Allah</td><td>Thr</td><td>Asn</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td>

<td>ΤΑΤ</td><td>ATG</td><td>GAT</td><td>Gct</td><td>GTT</td><td>GTT</td><td>AAT</td><td>TCA</td><td>AAA</td><td>TCT</td><td>TTG</td>

<td>Tyr</td><td>Met</td><td>Asp</td><td>Allah</td><td>Go</td><td>Go</td><td>Asn</td><td>To be</td><td>Lys</td><td>To be</td><td>Read</td>

<td></td><td> 125</td><td></td><td></td><td></td><td></td><td> 130</td><td></td><td></td><td></td><td> •</td>

<td>A CA</td><td>GAT</td><td>GTT</td><td>ATT</td><td>CAA</td><td>AAA</td><td>GTA</td><td>A CA</td><td>Gct</td><td>ATT</td><td>Gct</td>

<td>Thr</td><td>Asp</td><td>Go</td><td>Ile</td><td>Gin</td><td>Lys</td><td>Go</td><td>Thr</td><td>Allah</td><td>Ile</td><td>Allah</td>

<td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td><td> 145</td>

<td>ACT</td><td>GTT</td><td>TCT</td><td>AGT</td><td>Gcc</td><td>AAC</td><td>AAA</td><td>CAA</td><td>ATG</td><td>TTG</td><td>GAA</td>

<td>Thr</td><td>Go</td><td>To be</td><td>To be</td><td>Allah</td><td>Asn</td><td>Lys</td><td>Gin</td><td>Met</td><td>Read</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td>

<td>CAA</td><td>CAA</td><td>GAA</td><td>AAA</td><td>GAG</td><td>CAA</td><td>AAA</td><td>GAG</td><td>CTT</td><td>AGC</td><td>CAA</td>

<td>Gin</td><td>Gin</td><td>Glu</td><td>Lys</td><td>Glu</td><td>Gin</td><td>Lys</td><td>Glu</td><td>Read</td><td>To be</td><td>Gin</td>

<td></td><td></td><td></td><td> 160</td><td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td>

<td>THE AG</td><td>TCA</td><td>GAA</td><td>ACT</td><td>GTT</td><td>AAA</td><td>THE AG</td><td>AAC</td><td>TAC</td><td>AAC</td><td>CAG</td>

<td>Lys</td><td>To be</td><td>Glu</td><td>Thr</td><td>Go</td><td>Lys</td><td>Lys</td><td>Asn</td><td>Tyr</td><td>Asn</td><td>Gin</td>

<td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td><td></td><td></td>

<td>TTC</td><td>GTT</td><td>TCT</td><td>CTT</td><td>TCA</td><td>CAA</td><td>AGT</td><td>TTG</td><td>GAT</td><td>TCT</td><td>CAA</td>

<td>Phe</td><td>Go</td><td>To be</td><td>Read</td><td>To be</td><td>Gin</td><td>To be</td><td>Read</td><td>Asp</td><td>To be</td><td>Gin</td>

<td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td>

<td>Gct</td><td>CAA</td><td>GAA</td><td>TTG</td><td>ACT</td><td>TCA</td><td>CAA</td><td>CAA</td><td>Gct</td><td>GAA</td><td>CTC</td>

<td>Allah</td><td>Gin</td><td>Glu</td><td>Read</td><td>Thr</td><td>To be</td><td>Gin</td><td>Gin</td><td>Allah</td><td>Glu</td><td>Read</td>

<td> 190</td><td></td><td></td><td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td>

<td>AAA</td><td>GTT</td><td>GCG</td><td>ACT</td><td>TTG</td><td>AAC</td><td>TAT</td><td>CAA</td><td>GCA</td><td>A CA</td><td>ATT</td>

<td>Lys</td><td>Go</td><td>Allah</td><td>Thr</td><td>Read</td><td>Asn</td><td>Tyr</td><td>Gin</td><td>Allah</td><td>Thr</td><td>Ile</td>

<td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td><td> 210</td><td></td>

<td>GCA</td><td>ACT</td><td>GCG</td><td>CAA</td><td>GAT</td><td>AAA</td><td>AAA</td><td>CAA</td><td>Gct</td><td>TTA</td><td>TTA</td>

<td>Allah</td><td>Thr</td><td>Allah</td><td>Gin</td><td>Asp</td><td>Lys</td><td>Lys</td><td>Gin</td><td>Allah</td><td>Read</td><td>Read</td>

215

220

797

830

863

896

9

962

995

1028

1061

1094

<img file="PT97081B_D0076.tif" />

<img file="PT97081B_D0077.tif" />

<td>GAT</td><td>GAA</td><td>AAA</td><td>GCA</td><td>Gct</td><td>GCA</td><td>GAA</td><td>AAA</td><td>GCA</td><td>Gct</td><td>CAA</td>

<td>Asp</td><td>Glu</td><td>Lys</td><td>Allah</td><td>Allah</td><td>Allah</td><td>Glu</td><td>Lys</td><td>Allah</td><td>Allah</td><td>Gin</td>

<td></td><td></td><td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td>

<td>GAA</td><td>GCA</td><td>Gct</td><td>AAA</td><td>AAA</td><td>CAA</td><td>GCG</td><td>Gct</td><td>TAT</td><td>GAA</td><td>Gct</td>

<td>Glu</td><td>Allah</td><td>Allah</td><td>Lys</td><td>Lys</td><td>Gin</td><td>Allah</td><td>Allah</td><td>Tyr</td><td>Glu</td><td>Allah</td>

<td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td><td></td><td></td><td></td><td></td>

<td>CAA</td><td>CAA</td><td>AAA</td><td>GAA '</td><td>GCA</td><td>GCA</td><td>CAA</td><td>GCA</td><td>CAA</td><td>GCA</td><td>Gct</td>

<td>Gin</td><td>Gin</td><td>Lys</td><td>Glu</td><td>Allah</td><td>Allah</td><td>Gin</td><td>Allah</td><td>Gin</td><td>Allah</td><td>Allah</td>

<td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td>

<td>TCA</td><td>A CA</td><td>GCA</td><td>GCA</td><td>ACT</td><td>Gct</td><td>AAA</td><td>Gct</td><td>GTA</td><td>GAA</td><td>GCA</td>

<td>To be</td><td>Thr</td><td>Allah</td><td>Allah</td><td>Thr</td><td>Allah</td><td>Lys</td><td>Allah</td><td>Go</td><td>Glu</td><td>Allah</td>

<td></td><td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td>

<td>GCA</td><td>ACT</td><td>TCA</td><td>TCA</td><td>Gct</td><td>TCT</td><td>Gct</td><td>TCA</td><td>TCT</td><td>AGT</td><td>CAA</td>

<td>Allah</td><td>Thr</td><td>To be</td><td>To be</td><td>Allah</td><td>To be</td><td>Allah</td><td>To be</td><td>To be</td><td>To be</td><td>Gin</td>

<td></td><td></td><td></td><td> 270</td><td></td><td></td><td></td><td></td><td> 275</td><td></td><td></td>

<td>Gct</td><td>CCA</td><td>CAA</td><td>GTA</td><td>AGT</td><td>A CA</td><td>AGC</td><td>ACT</td><td>GAT</td><td>AAT</td><td>A CA</td>

<td>Allah</td><td>Pro</td><td>Gin</td><td>Go</td><td>To be</td><td>Thr</td><td>To be</td><td>Thr</td><td>Asp</td><td>Asn</td><td>Thr</td>

<td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>A CA</td><td>TCA</td><td>AAT</td><td>Gct</td><td>AGT</td><td>Gcc</td><td>TCA</td><td>AAC</td><td>AGT</td><td>TCT</td><td>AAT</td>

<td>Thr</td><td>To be</td><td>Asn</td><td>Allah</td><td>To be</td><td>Allah</td><td>To be</td><td>Asn</td><td>To be</td><td>To be</td><td>Asn</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td>

<td>AGT</td><td>TCA</td><td>TCA</td><td>AAC</td><td>TCA</td><td>AGT</td><td>TCA</td><td>AGT</td><td>TCT</td><td>AGC</td><td>AGT</td>

<td>To be</td><td>To be</td><td>To be</td><td>Asn</td><td>To be</td><td>To be</td><td>To be</td><td>To be</td><td>To be</td><td>To be</td><td>To be</td>

<td> 300</td><td></td><td></td><td></td><td></td><td> 305</td><td></td><td></td><td></td><td></td><td> 310</td>

<td>TCA</td><td>TCA</td><td>AGC</td><td>TCA</td><td>AGC</td><td>TCA</td><td>AGC</td><td>TCA</td><td>AGT</td><td>AAT</td><td>TCT</td>

<td>To be</td><td>To be</td><td>To be</td><td>To be</td><td>To be</td><td>To be</td><td>To be</td><td>To be</td><td>To be</td><td>Asn</td><td>To be</td>

<td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td><td></td>

<td>AAT</td><td>Gct</td><td>GGT</td><td>GGG</td><td>AAT</td><td>A CA</td><td>AAT</td><td>TCA</td><td>GGC</td><td>ACT</td><td>AGT</td>

<td>Asn</td><td>Allah</td><td>Gly</td><td>Gly</td><td>Asn</td><td>Thr</td><td>Asn</td><td>To be</td><td>Gly</td><td>Thr</td><td>To be</td>

<td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td>

<td>ACT</td><td>Gga</td><td>AAT</td><td>ACT</td><td>Gga</td><td>Gga</td><td>A CA</td><td>ACT</td><td>ACT</td><td>GGT</td><td>GGT</td>

<td>Thr</td><td>Gly</td><td>Asn</td><td>Thr</td><td>Gly</td><td>Gly</td><td>Thr</td><td>Thr</td><td>Thr</td><td>Gly</td><td>Gly</td>

335 340

1127

1160

1193

1226

1259

1292

1325

1358

1391

1424

<img file="PT97081B_D0078.tif" />

<td>AGC</td><td colspan="7">GGT ATA AAT AGT TCA CCA ATT</td>

<td>To be</td><td>Gly</td><td>Ile</td><td>Asn</td><td>To be</td><td>To be</td><td>Pro</td><td>Ile</td>

<td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td>

<td>TAT</td><td>Gct</td><td>GTT</td><td>GGT</td><td>Gga</td><td>TGT</td><td>ACT</td><td>GAC</td>

<td>Tyr</td><td>Allah</td><td>Go</td><td>Gly</td><td>Gly</td><td>Cys</td><td>Thr</td><td>Asp</td>

<td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td>

<td>CAA</td><td>TAC</td><td>TTT</td><td>Gct</td><td>GCA</td><td>CAA</td><td>Gga</td><td>ATT</td>

<td>Gin</td><td>Tyr</td><td>Phe</td><td>Allah</td><td>Allah</td><td>Gin</td><td>Gly</td><td>Ile</td>

<td></td><td></td><td></td><td></td><td> 370</td><td></td><td></td><td></td>

<td>AAT</td><td>ATC</td><td>ATG</td><td>CCT</td><td>GGT</td><td>AAT</td><td>GGT</td><td>Gga</td>

<td>Asn</td><td>Ile</td><td>Met</td><td>Pro</td><td>Gly</td><td>Asn</td><td>Gly</td><td>Gly</td>

<td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>TCT</td><td>AAT</td><td>Gga</td><td>CCT</td><td>Gcc</td><td>CAA</td><td>GGC</td><td>GTG</td>

<td>To be</td><td>Asn</td><td>Gly</td><td>Pro</td><td>Allah</td><td>Gin</td><td>Gly</td><td>Go</td>

<td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td>

<td>GTA</td><td>Gga</td><td>Gct</td><td>Gct</td><td>CCT</td><td>GGT</td><td>GTT</td><td>ATC</td>

<td>Go</td><td>Gly</td><td>Allah</td><td>Allah</td><td>Pro</td><td>Gly</td><td>Go</td><td>Ile</td>

<td></td><td> 400</td><td></td><td></td><td></td><td></td><td> 405</td><td></td>

<td>TTC</td><td>TCA</td><td>Gct</td><td>GAT</td><td>TTT</td><td>GTT</td><td>Gga</td><td>TAT</td>

<td>Phe</td><td>To be</td><td>Allah</td><td>Asp</td><td>Phe</td><td>Go</td><td>Gly</td><td>Tyr</td>

<td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td><td></td>

<td>CCT</td><td>TAC</td><td>GGT</td><td>CAC</td><td>GTA</td><td>Gct</td><td>ATT</td><td>GTA</td>

<td>Pro</td><td>Tyr</td><td>Gly</td><td>His</td><td>Go</td><td>Allah</td><td>Ile</td><td>Go</td>

425

GGA AAT CCT 1457

Gly Asn Pro

ΤΑΤ GTA TGG 1490

Tyr Vai Trp

365

TAT ATC AGA 1523

Tyr Ile Arg

375

CAA TGG GCT 1556

Gin Trp Wing

385

CTC CAT GTT 1589

Read his go

GCA TCA AGC 1622

Ala Ser Ser

GCA AAC TCA 1655

Wing Asn Ser

420

AAA TCA GTT 1688

Lys Ser Vai

430

<img file="PT97081B_D0079.tif" />

<td>AAT</td><td>TCA</td><td>GAT</td><td>GGT</td><td>A CA</td><td>ATT</td><td>ACT</td><td>ATC</td><td>AAA</td><td>GAA</td><td>GGC</td><td> 1721</td>

<td>Asn</td><td>To be</td><td>Asp</td><td>Gly</td><td>Thr</td><td>ile</td><td>Thr</td><td>Ile</td><td>Lys</td><td>Glu</td><td>Gly</td><td></td>

<td></td><td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td>

<td>Gga</td><td>TAT</td><td>GGT</td><td>A CA</td><td>ACT</td><td>TGG</td><td>TGG</td><td>Gga</td><td>CAT</td><td>GAA</td><td>CGT</td><td> 1754</td>

<td>Gly</td><td>Tyr</td><td>Gly</td><td>Thr</td><td>Thr</td><td>Trp</td><td>Trp</td><td>Gly</td><td>His</td><td>Glu</td><td>Arg</td><td></td>

<td></td><td></td><td> 445</td><td></td><td></td><td></td><td></td><td> 450</td><td></td><td></td><td></td><td></td>

<td>ACT</td><td>GTA</td><td>AGT</td><td>GCG</td><td>TCT</td><td>GGT</td><td>GTT</td><td>ACT</td><td>TTC</td><td>TTG</td><td>ATG</td><td> 1787</td>

<td>Thr</td><td>Go</td><td>To be</td><td>Allah</td><td>To be</td><td>Gly</td><td>Go</td><td>Thr</td><td>Phe</td><td>Read</td><td>Met</td><td></td>

<td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td><td></td>

CCA AAC TAG AAAAAAGTCT TAATAAATAA AAAATAGTGG 182 6

Pro asn

O ⁴⁶⁵

TTTGATAGTG GGGAATAATT TTCCTTCTGT CAAATCATTT 1866

TTTATTATTG TGGTATAATA ATAAGGAAAA ATGATAAGGG 1906

AATG GATAGATACA

1920

<img file="PT97081B_D0080.tif" />

SEQ ID NO: 2

TYPE OF SEQUENCE: Nucleotides with corresponding polypeptide STRING LENGTH '279 bp TYPE OF TWO CHAINS TOPOLOGY? linear

Recombinant Molecule Type

Hirudo

ORIGINAL SOURCE ORGANIZATION L lactis LM0230

IMMEDIATE EXPERIMENTAL SOURCE? PUCRS plasmid (DSM 5303 ™, pia deo pML310 (see European patent application EP-A-1634))

5BlackCharacteristics;

the al bp Mst bp signal peptide coding region of the disulfide-hi rud i na

PROPERTIES? DNA fusion of L.1 act isLMããna encoding MSP signal peptide and hirudin structural gene for bacterial secreted hirudin production »

<td>ATG</td><td>AAA</td><td>AAA</td><td>THE AG</td><td>ATT</td><td>ATC</td><td>TCA</td><td>Gct</td><td>ATT</td><td>TTA</td><td>ATG</td><td>TCT</td><td> 36</td>

<td>Met</td><td>Lys</td><td>Lys</td><td>Lys</td><td>Ile</td><td>Ile</td><td>To be</td><td>Allah</td><td>Ile</td><td>Read</td><td>Met</td><td>To be</td><td></td>

<td></td><td></td><td> -25</td><td></td><td></td><td></td><td></td><td> -20</td><td></td><td></td><td></td><td></td><td></td>

<td>A CA</td><td>GTG</td><td>ATA</td><td>CTT</td><td>TCT</td><td>Gct</td><td>GCA</td><td>Gcc</td><td>CCG</td><td>TTG</td><td>TCA</td><td>GGT</td><td> 72</td>

<td>Thr</td><td>Go</td><td>Ile</td><td>Read</td><td>To be</td><td>Allah</td><td>Allah</td><td>Allah</td><td>Pro</td><td>Read</td><td>To be</td><td>Gly</td><td></td>

<td> -15</td><td></td><td></td><td></td><td></td><td> -10</td><td></td><td></td><td></td><td></td><td> -5</td><td></td><td></td>

<td>GTT</td><td>TAC</td><td>Gct</td><td>GTT</td><td>GTT</td><td>TAC</td><td>ACC</td><td>GAC</td><td>TGC</td><td>ACC</td><td>GAA</td><td>TCT</td><td> 108</td>

<td>Go</td><td>Tyr</td><td>Allah</td><td>Go</td><td>Go</td><td>Tyr</td><td>Thr</td><td>Asp</td><td>Cys</td><td>Thr</td><td>Glu</td><td>To be</td><td></td>

<img file="PT97081B_D0081.tif" />

<td rowspan="3"></td><td rowspan="3">GGT Gly 10</td><td colspan="2">CAG AAC</td><td colspan="9">CTG TGC CTG TGC GAA GGT TCT AAC GTT</td><td rowspan="3"> 144</td>

<td rowspan="2">Gin</td><td rowspan="2">Asn</td><td rowspan="2">Read</td><td rowspan="2">Cys</td><td colspan="6">Read Cys Glu Gly Ser Asn</td><td rowspan="2">Go</td>

<td colspan="2"> 15</td><td colspan="4"> 20</td>

<td></td><td>TGC</td><td>GGT</td><td>CAG</td><td>GGT</td><td>AAC</td><td>AAA</td><td>TGC</td><td>ATC</td><td>CTG</td><td>GGT</td><td>TCT</td><td>GAC</td><td> 180</td>

<td>J</td><td>Cys</td><td>Gly</td><td>Gin</td><td>Gly . 25</td><td>Asn</td><td>Lys</td><td>Cys</td><td>Ile</td><td>Read 30</td><td>Gly</td><td>To be</td><td>Asp</td><td></td>

<td></td><td>GGT</td><td>GAA</td><td>AAA</td><td>AAC</td><td>CAG</td><td>TGC</td><td>GTT</td><td>ACC</td><td>GGC</td><td>GAA</td><td>GGT</td><td>ACC</td><td> 216</td>

<td></td><td>Gly</td><td>Glu</td><td>Lys</td><td>Asn</td><td>Gin</td><td>Cys</td><td>Go</td><td>Thr</td><td>Gly</td><td>Glu</td><td>Gly</td><td>Thr</td><td></td>

40 45

<td rowspan="2">CCG Pro</td><td colspan="4">AAA CCG CAG TCT</td><td colspan="7">CAC AAC GAC GGT GAC TTC GAA</td><td rowspan="2"> 252</td>

<td>Lys</td><td>Pro</td><td>Gin</td><td>To be 50</td><td>His</td><td colspan="2">Asn asp</td><td>Gly</td><td>Asp 55</td><td>Phe</td><td>Glu</td>

<td>GAA</td><td>ATC</td><td>CCG</td><td>GAA</td><td>GAA</td><td>TAC</td><td>CTG</td><td>CAG</td><td>Tag</td><td></td><td></td><td></td><td> 279</td>

<td>Glu</td><td>Ile</td><td>Pro</td><td>Glu</td><td>Gin</td><td>Tyr</td><td>Read</td><td>Gin</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td><td> 65</td><td></td><td></td><td></td><td></td><td></td>

<img file="PT97081B_D0082.tif" />

<img file="PT97081B_D0083.tif" />

comprising:

)

(b) the EcoRI / SalI insert of the pUCF plasmid. approximately 3.0 kbp L or a functional fragment thereof, or a DNA sequence which hybridizes to said functional insert or gum. or comprises a promoter region that is operably linked to bibrid DNA, or