Process for producing bacterial vectors

Although genetic engineering knows many prokaryotic vector host systems in which homologous or heterologous genes can be encoded, there is a continuing need for new systems that are more advantageous than those known in the art.

Most results of DNA manipulation have been achieved with bacteria such as Escherichia coli or Bacillus subtilis, although the industrial importance of lactic acid bacteria

<td>much larger.</td><td>Because of this, many efforts have already been made with lactic acid.</td>

<td>bacteria,</td><td>especially Lactobaci11us or Lactococcus fa-</td>

<td>well, homolog</td><td>or to clone and express heterologous genes</td>

capable of generating vectors [e.g. PCT WO 85/03945; Gasson and Anderson (1985), ΕΡ-Ά-0 316 677;

<td>patent description;</td><td>Bates et al., 1989; boss and co-worker</td>

received (1985); or Chassy (1987) and De Vos (1987). Lactococcus species were previously called Streptococcus species.

The Lactococcus strains examined so far carry a typical Ρ1 plasmid preparation consisting of several different plasmids; this property may also serve as a basis for strain discrimination (Davies et al., 1981). For genetic studies, plasmid-free strains were also generated by repeated healing of plasmid function (De Vos (1987)). For example, the ρ1 plasmid set of strains of L. lactis 712 (also known as LL712) consists of a plasmid:

Md molecular weight pSH71, a 2.5

Md os pSH72.

pSH73, 5.2 Md, pSH74, 9 Md, and 33

Starting from PWV01 • · · ρ1 plasmids (Otto et al., 1982) created various cloning vectors. In the construction of vectors, either marker genes (e.g., antibiotic resistance genes) were inserted into plasmids, or a replication origin (capable of providing replication of a particular DNA fragment) was searched for and inserted into a DNA construct. Recently, plasmid pNZ12 was constructed, which contains the replication origin of pSH71 in the form of a 1.7 kbp Clal restriction fragment (Gasson and Anderson (1985)). The replication origin of pSH71 is functional in both Gram-positive (e.g., Bacillus species) and Gram-negative (e.g., E. coli) bacteria.

Starting from these plasmids, cloning vectors capable of introducing homologous or heterologous genes into and expressing homologous or heterologous genes have been created. The vectors have been used to generate transformant Lactoccus strains with better properties than those known in the food and feed industry; for example, bacteriophage resistant (ΕΡ-Ά-0 316 677) or bovine prochymosin (PCT WO 85/03945) L. lactis strains. The importance of vectors cloning the products of homologous or heterologous genes is, however, not only that they can be used to create more lactic acid bacterial strains, but also that: · · · · ····· · · · · · · · · · · · · · · · · · · · · · · ·

- We can produce 4 recombinant proteins. One of the major problems with the production of heterologous proteins by microorganisms is the purification of the product. Purification of intracellular proteins can be accomplished with poor yield. making it much simpler.

time-consuming and often only yes but secreted products are purified The problems can be avoided by preferably using vector-host systems for the production of recombinant proteins that select it in the culture medium.

Another advantage of secreted proteins is that they appear in a native, biologically active conformation, so that conformational restoration procedures such as those used to purify intracellular proteins do not have to be applied.

Secretion of proteins generally requires a signaling peptide that binds to the N-terminus of the primary translation product and ensures that the protein passes through the secretory pathway. It is preferable that the tagging peptide is cleaved enzymatically through the membrane via the membrane, but this is not always the case.

Object and embodiments of the invention

The present invention relates to novel hybrid vectors, all of which

They are capable of replication in Gram-negative and Gram-positive bacteria and / or express homologous or heterologous genes.

• · • « · · • ·

-The product of which - a stable protein - is secreted into the nutrient medium.

Embodiments of the present invention are, in particular, novel hybrid vectors comprising novel DNA inserts carrying a promoter region and a DNA sequence comprising a coding sequence for a signaling peptide and a gene encoding a polypeptide which is hitherto unknown. product of L. the protein most abundant in lactis culture is precipitation of the medium with trichloroacetic acid, separation of the precipitated proteins by electrophoresis1 on a SDS-polyacrylamide gel and staining of the gel with Coomassie blue; this product is hereinafter referred to as MSP (Major Secretion Product).

Further embodiments of the present invention are novel hybrid vectors comprising an origin of replication from a 2.5 Md molecular weight plasmid of L. lactis LL712 or a replication origin thereof operable in both Gram-positive and Gram-negative bacteria. .

Accordingly, embodiments of the present invention also provide novel hybrid vectors which have the promoter region of the MSP gene or a similar gene. the coding DNA sequence of the tag peptide and / or the gene

<img file="HUT57263A_D0001.tif" />

and the origin of replication of L. lactis LL712 2.5 Md or 5.2 Md or a similar origin of replication.

The present invention also encompasses functional fragments of novel DNA inserts or origin of replication per se. These can be used to generate novel expression vectors for the production and secretion of homologous or heterologous proteins in lactic acid bacterial hosts.

The invention further relates to a method for the production of novel DNA molecules and hybrid vectors, and to a method for producing secreted gene products using hosts transformed with the novel hybrid vectors of the invention.

The invention also relates to purified MSP protein.

Detailed Description of the Invention

DNA mo1ekulák

The present invention relates to hybrid vectors which

a) Approx. 3.5 kbp, of L. lactis origin

An EcoRI / SalI insert or a functional fragment thereof, or

b) a DSN sequence capable of hybridizing to the above insert or a functional fragment thereof. or a promoter region naturally operably linked to the hybridizing DNA sequence, or

(c) a degenerate DNA sequence of (a) or (b) encoding a signaling peptide; or

d) a derivative of a DNA molecule according to (a), (b) or (c) and / or (I) a 2.5 Md plasmid of L. lactis LL712;

<td>or (II)</td><td>L. lactis LL712 has a 5.2 Md molecular weight plasmid.</td>

<td>midjának,</td><td>or (III) ab. lactis LL712 or 2.5 Md</td>

<td>5.2 Md</td><td>p1 plasmids1 have the same incompatibility tube-</td>

The plasmid belonging to the harboring a plasmid belonging to port 1 is repainted with the origin of replication.

Plasmid pUCRS has an approx. The 3.5 kbp EcoRI / SalI insert from L. lactis contains the promoter region of the MSP gene, the coding sequence of the MSP signaling peptide, and the coding region of MSP. MSP carrying the signaling peptide is hereinafter referred to as pre-MSP. The EcoRI / SalI insert contains the DNA sequence of SEQ ID No. 1, which is 1920 bp long and is numbered so that base 1 is closer to the EcoRI restriction site.

In SEQ ID NO: 1, the coding sequence for MSP is described in pages 492-1793. between bases. The 27 peptide coding peptide codes have the DNA sequence from the end of the promoter sequence to the coding region of the mature MSP, i.e., from base 491 to «·» · * ·

411th base. The marker peptide of MSP or a functional fragment thereof ensures that the host cell, such as a lactic acid bacterium such as Lactococcus letet. or a

<td>Baci1lus species. sample</td><td>Bacillus thuringiensis, secretes a</td>

<td>protein when the marker</td><td>peptide C-terminal amino acid is covalent</td>

<td>bonding</td><td>to the N-terminal amino acid of the protein. The</td>

The covalent attachment of the C-terminal amino acid of the MSP tag peptide to the N-terminal amino acid of the host cell secreted protein is achieved by constructing a fusion gene comprising a C-terminal encoding the entire MSP tag peptide or a functional fragment thereof. The DNA sequence is located in a suitable reading frame, linked to the 5 'end of the structural gene of the desired protein. An example of such a fusion gene is SEQ ID No. 2. A DNA sequence constructed from the coding region of the MSP signaling peptide and the structural gene of hirudin.

The marker peptide of MSP and the amino acid sequence of MSP are also shown in SEQ ID No. 1.

The primer site of the MSP gene is upstream of the DNA sequence encoding the MSP signaling peptide and

It consists of 1500 nucleotides, preferably from 100 to 1000 nucleotides. The approx. In the 3.5 kbp EcoRI / SalI insert, this promoter region is located 1 upstream of the EcoRI site.

from base 411 to base 411. More specifically, the ···· »* 4« ·· · * 44 · * · 4 * · 4 ·· • 4 · 4 · * * · ·· »* · r 444« «Γ 99

- 9 promoter regions from the first HindIII restriction site from base 1 to base 411. The promoter region is capable of binding to RNA polymerase or regulatory proteins and thereby influencing the expression of the associated structural gene.

The term functional fragment refers to DNA fragments that retain their promoter, labeling, and / or structural functions.

Preferred are functional fragments comprising the promoter region of MSP, the coding sequence of the MSP signaling peptide, or the promoter region and the coding sequence of the signaling peptide. However, the fragments of the invention may also be DNA sequences encoding smaller peptides that are capable of retaining and / or signaling the function of the MSP promoter.

For example, fragments with promoter function are DNA sequences from the Lactis 3.5 kbp Eco RI / Sal I insert, more particularly from the HindIII restriction site to SEQ ID NO: 1, base 410. Similarly, fragments with promoter function are all DNA sequences that span from any base between EcoRI and HindIII restriction sites to base 410 of SEQ ID NO: 1. The shorter fragments of the promoter region also retain their promoter activation.

• · · ·

<img file="HUT57263A_D0002.tif" />

A DNA fragment encoding a flag peptide is, for example, the sequence from base 411 to base 491 of SEQ ID NO: 1. The fragment encoding the tag peptide may extend from the 5 'end into the promoter region from which it may carry a region that no longer retains promoter function.

The fragment containing the promoter function of the MSP and the coding region of the tagging peptide may span from the EcoRI restriction site of the insert to base 491 of SEQ ID NO: 1. Another fragment that retains both functions is the sequence from the first Hindi II restriction site to base 491, while other such fragments are those of EcoRI and

They start from any base between Hindi II restriction sites and go to base 491 of SEQ ID NO: 1.

Said fragments may also contain linkers, i.e., fragments which provide for their attachment to other DNA molecules. Suitable linkers are DNA sequences that are capable of mapping to the restriction sites of the DNA sequences to be linked. DNA fragments may also contain specific restriction sites.

DNA molecules capable of hybridizing with an insert are those that hybridize to them under normal conditions; a conventional hybridization procedure is, for example, that of Benton and Davis (1977). Such hybridizing DNA molecules may contain, for example, MSPs · · · · as hybridizing sequences.

<img file="HUT57263A_D0003.tif" />

different sequence variants of structural genes: such sequence variants are hereinafter referred to as

It has a MSP gene

Accordingly, which is operably linked to DNA sequences capable of hybridizing with the insert,

It is also the promoter region of genes structurally related to the MSP gene.

For example, genes structurally related to the MSP gene are L.

Lactobacillus or Lactococcus species, such as L. crernor is or L. thermophilus. Also naturally occurring variants are those encoded by an isogen of the L. lactis chromosome or an isogen of a natural plasmid.

DNA molecules containing a gene related to the MSP gene can be isolated in a conventional manner.

The DNA molecules of the invention also contain degenerate DNA sequences. Their degeneracy within the genetic code is understood to mean that an unlimited number of their nucleotides can be replaced by other nucleotides without undergoing a change in the amino acid sequence they encode. DNA sequences degenerated in this way can be very useful because they have different restriction sites or carry the codons preferred by the particular host. DNA encoding the MSP flag peptide or a variant thereof is preferred. · · · · · · · · · · · · · · · · · · · · · ·

Degenerate variants of 12 sequences.

When used in reference to the DNA molecules described in a), b) or c), we mean fragments, mutants or larger derivatives thereof. Preferred are fragments which retain their promoter, signaling, or structural function, examples of which have been mentioned previously.

A mutant of a DNA molecule according to a), b) or c) is a molecule that has been altered by deletion, insertion, inversion or point mutation. Mutations can be natural or artificial; the latter can be generated in vitro or in vivo by conventional methods. The restriction pattern of the mutants may differ from the parent molecule.

Larger derivatives of the DNA molecules of a), b) or c) may be cleaved, for example, from the L. lactis genome. Such are found in the L. lactis LM0230 gene library, which is fragmented by nucleic acids with appropriate restriction enzymes - e.g., Sau3AI. By treatment with EcoRI, BamHI or Hindi II, by ligation to appropriate vectors, such as lambdaEMBL3 or the pBR322 plasmid, E. coli and re-digested with the same or other restriction enzymes.

That) . b) overlapping recombinant DNA molecules themselves into larger derivatives of the DNB molecules of (c), e.g., containing appropriate restriction sites

Linker1 or regulatory sequences, e.g., a promoter, a coding sequence for a signaling peptide, or a terminator1 and an appropriate reading frame.

I, or a ΐ sequence from a vector, such as a phage or plasmid used to make a hybrid vector. The overlapping sequences of the larger derivatives are a; genes can be promoted.

i

L. lactis LL712 has a molecular weight of 2.5 Md or 5.2 Md

^! Replication origins from P1 plasmids or other plasmids belonging to the same incompatibility group allow a plasmid in lactic acid bacteria such as Lactococcus species (such as L. lactis, L. lactis).

Diacetylactis or L. cremoris), E. coli or other Gram-negative bacteria or non-lactic bacteria

/ Grarn-positive bacteria, such as Baci lus species (such as B. thuringiensis), are also replicated.

L. lactis LL712 has a molecular weight of 2.5 Md or 5.2 Md

For example, replication origin sites from P1 plasmids or other plasmids belonging to the same incompatibility group may include the complete linearized plasmid obtained by treatment with appropriate restriction endonucleases. Accordingly, the hybrid vectors of the invention

<img file="HUT57263A_D0004.tif" />

- 14 ci may also contain the complete DNA sequence of plasmids having a molecular weight of 2.5 Md or 5.2 Md or other plasmids belonging to the same incompatibility group. For example, both plasmids can be isolated from the L. lactis strain LL712 deposited under accession number DSM 5804 with the Deutsche Sammlung von Mikroorganismén und Zellkulturen.

DNA sequences that function as the origin of replication may also be fragments of said plasmids that retain their function of origin of replication. For example, such fragments may be obtained by fragmenting the appropriate plasmid or ρ1 plasmid fragment, for example, by physical methods, chemical methods, or DNA-degrading enzymes (such as Bal31 or S1 endonucleases, exonuclease III, or restriction endonucleases) and isolating the appropriate sequence. Such fragments may be, in particular, restriction fragments which are digested with one or two different restriction endonucleases (e.g., Ndel, SphI and / or EcoRV for 2.5 Mdos, for example, Sau3A, Ndel for 5.2 Md). , HindIII, AccI and / or EcoRI).

For example, the 2.5 Md plasmid has an origin of replication at ca. 1.8 kbp on the NdeI / SphI fragment; 3.1 kbp EcoRV / Sphl fragment (obtained by partial digestion of the 3.5 kbp Sphl fragment with EcoRV), the approx. On a 3.5 kbp Sphl fragment. the app.

1.2 kbp Ndel / EcoRV fragment or the approx. It is located on a 3.5 kbp EcoRV / SphI fragment (obtained by partial digestion of the Sphl fragment of about 3.5 kbp with EcoRV). The approx. The 3.5 kbp SphI and the other fragments mentioned above are all part of the plasmid pSC12. Construction of plasmid pSC12 is described in section 1.2. described in Example 4; a restriction map is shown in Figure 2.

For example, the 5.2 Md plasmid has an origin of replication at ca. On a 1.0 kbp NdeI / HindIII fragment, the approx. On a 2.2 kbp NdeI / AccI fragment, the approx. 2.7 kbp NdeI / Eco RI fragment, the approx. 3.1 kbp on the NdeI / / Sau3A fragment; On a 2.0 kbp Sau3A / HindIII fragment, the approx. On a 3.2 kbp Sau3A / AccI fragment; 3.7 kbp Sau3A / 'Eco RI fragment or the approx. It is located on a 4.1 kbp Sau3A fragment. Fragments having the Sau3A end can be prepared by partial digestion of the 5.2 Md plasmid (pSC18) with Sau3A or by partial digestion of the corresponding fragments of the plasmid. Construction of plasmid pSC18 is described in Section 1.3. described in Example 4; a restriction map is shown in Figure 3.

Plasmids of the same incompatibility group cannot coexist permanently in a host cell. The origin of replication of such plasmids is similar to each other.

« ···

<img file="HUT57263A_D0005.tif" />

A recombinant DNA molecule may comprise, for example, linkers and other sequences from a vector, such as a phage or plasmid, and optionally a marker gene, such as a resistance gene (e.g., an erythromycin, ampicillin or tetracycline resistance gene or a similar gene). A recombinant DNA molecule containing an origin of replication according to the invention may be, for example, a vector plasmid of lactic acid bacteria. preferably, a commuting vector which is capable of replication in at least lactic acid bacteria and E. coli and which contains a single Lactococcus origin of replication origin of the invention. Preferred commuter vectors are pSC12, pSC12deltaP, pSC12deltaN, pSC12deltaNP, pSC18, pSC18deltaP or pSC18deltaN. Preferred shuttle vectors for expressing foreign genes in lactic acid bacteria are pSC12HIR, pSC12HIR-1 or pSC12HIRTerm. Preferred commuter vectors are described in the Examples.

A DNA sequence that can function as an origin of replication at least in lactic acid bacteria and in E. coli that satisfies the conditions of (e) may also be an inversion, insertion, deletion, or point mutation sequence. which retained the replication origin function. In mutants, the nucleotide sequence of overlapping regions outside the true replication site may be altered, for example, by a point mutation (which is a · ···

<img file="HUT57263A_D0006.tif" />

f restriction sites) or by deletion such as the L. lactis insert of plasmids pSC12deltaP, pSC12deltaN, pSC12delt.aNP, pSC18deltaP or pSC18deltaN. By this true origin of replication is meant the smallest DNA fragment that can still function as an origin of replication.

The hybrid vectors of the invention are suitable for cloning in hosts, such as fungi or especially bacteria, and can be generated from any vector useful in the field of gene manipulation, such as phages, cosmids or plasmids. They may be derivatives of, for example, lambda phages (NM989 or EMBL3), M13 broad (M13mpl8 or M13mpl9) or bacterial plasmids (pBR322, pUC18 or pUC19), but may be prepared from lactic acid or yeast plasmids (e.g., yeast or plasmid 2u). the presence of a plasmid from plasmids; in the latter case, an auxiliary phage is either necessary for the multiplication of the detective broad or the plasmid.

The hybrid vectors of the invention are a), b), c) or

(d) contain the nucleotide sequence of the DNA molecules corresponding to (d). They contain in particular approx. 3.5 kbp EcoRI / SalI insert from L. lactis or a fragment thereof carrying the MSP-tagging peptide and / or promoter. The DNA molecule of the invention inserted into the hybrid vector is t

Depending on its type, the vector may also contain a hybrid regulatory sequence. Such hybrid regulatory sequences may consist, in part, of DNA molecules corresponding to (a), (b), or (c), and partly other than those described therein. For example, a hybrid vector may contain the MSP promoter in combination with a DNA sequence encoding a signaling peptide other than MSP, or the MSP promoter.

A class of hybrid vectors other than MSP (so E.

DNA peptide encoding its marker peptide in combination with the promoter. Preferably, the transcription terminator of the present invention may also comprise a trpA terminator of a transcription terminator.

Hybrid vectors containing the coding DNA sequence of a signaling peptide of the invention, derived from the pre-MSP gene or a similar gene, are capable of producing secreted gene products in lactic acid bacteria, particularly in Lactococcus species, preferably L. lactis or

L. cremoris or other bacteria such as Bacillus species such as B. thuringiensis. Such vectors also contain a promoter capable of working in that bacterium, such as the MSP promoter in the case of lactic acid bacteria and optionally a transcriptional terminator that is operable in that bacterium. The use of a terminator can enhance the production of a recombinant binary gene product when it is operably linked to the corresponding homologous or heterologous structural gene; for example, if E. coli trpA terminator is operatively coupled to MSP • · «· f

• ·

Under the control of its 19 promoters, the production of desulphohirudin can be significantly enhanced by the hirudin gene expressed in acetic acid bacterial unions, encoding the DNA fragment encoding the MSP signaling peptide.

The hybrid vectors of the invention also contain a functional origin of replication in a particular host cell, such as lactic acid bacteria and / or Bacillus species and / or E. coli.

A DNA sequence carrying at least a functional origin of replication in lactic acid bacteria, such as the 1.7 kbp Clal fragment of the plasmid pSH71 of lactococci (Gasson and Anderson (1985)) or one of the DNA fragments of L. lactis LL712

It can be obtained from its 2.5 Md or 5.2 Md plasmids.

The hybrid vectors of the invention may also contain selectable markers; the choice of markers will depend on the host to be transformed, selected and cloned. Any selection marker may be appropriate if its phenotypic appearance may facilitate the selection of transformants. Antibiotic resistance markers, such as VH, are particularly suitable. ampicillin or tetracycline resistance, or, in the case of auxotrophic mutants, genes that compensate for the lack of a host cell, for example in the lactose metabolism.

<img file="HUT57263A_D0007.tif" />

Preferred embodiments of the hybrid vectors of the present invention are hybrid vectors comprising a homologous or heterologous structural gene, such as the MSP gene, or in particular a heterologous structural gene as defined above, which gene is operably integrated into the DNA sequence encoding a suitable MSP signaling peptide. which is regulated by the MSP promoter or a heterologous promoter.

Structural genes may be derived from viruses, prokaryotic or eukaryotic cells; They can be obtained from genomic DNA, cDNA from mRNA, or by chemical synthesis. They may encode a wide variety of useful polypeptides, including glycosylated polypeptides, in particular higher polypeptides of higher eukaryotes, preferably mammals, such as animals, but especially humans, such as enzymes which are useful in the food industry or in chemical synthesis or in human and animal diseases. or preventive polypeptides such as hormones, immunomodulatory polypeptides, antiviral and antitumor polypeptides, antibodies, antiviral antigens, vaccines, coagulation factors, nutrients and the like.

Examples of such heterogeneous genes include hormones (e.g., secretin, thyrcine, relaxin, calcitonin, luteinizing). · ···· ·· · · hormone parathyroid hormone, adrenocort icotropic i nt. rnelanocyte-stimulating hormone, 0-1 lipotropin, urogastrone or insulin), growth factors (e.g. growth factor or transforming growth factor [TGF0]), growth hormones (e.g. human or bovine growth factor, interleukin [interleukin-1 or -2], human macrophage migration inhibitory factor [MIF]), interferons (human α-interferons such as ccA, ccB, ctD, ctF, 13 or gamma interferons, or hybrid interferons such as interferon α-αD or β-αD, in particular BDBB hybrid interferon ), proteinase inhibitors (e.g., antitrypsin, SPLI, and the like), hepatitis virus antigens (e.g., hepatitis B virus surface and coat antigen, hepatitis A virus antigen, or hepatitis nonA-nonB) . ρ1-azminogen activators (e.g., tissue ρ1-aminogen activator or urokinase), tumor necrosis factor, somatostatin, renin, O-endorphin, immunoglobulins (e.g., light and / or heavy chains of immunoglobulin D, -E or -G) , or human-mouse hybrid immunoglobulins), immunoglobulin binding factors (e.g., immunoglobulin iNE binding factor [sCD23], calcitonin-like epitec, blood coagulant activity (p e d). Factor 1 — IX or Hg

<img file="HUT57263A_D0008.tif" />

I • · · · • ···· · · · · · · · · · · · · · · · · · · · · · · · · · ·

-VIIIc), erythropoietin, eglin (e.g. eglin-C). hirudin, desulfo-hirudin (e.g., HV1, HV2, HV3, [Lys^&lt;v] -HV2, [Leu¹.Thr²] -HV1 or PA desulphohirudin variants).

structural genes encoding human superoxide dismutase, viral thymidine kinase, β-lactamase or glucose isomerase.

The gene encoding HV1 desulfo-hirudin is preferred. In the hybrid vectors of the invention, the DNA molecule encoding the promoter and / or the MSP signaling peptide is operably linked to the structural gene of the polypeptide so as to ensure efficient expression of the polypeptide.

Preferred hybrid vectors are pUCRS, M13mpl8RS, M13mpl9H, pVAHIR, pVAHIR-1, pSC12HIR and especially pSC12HIRTerm; hybrid vectors are described in the Examples.

The invention further relates to a DNA molecule which is in itself

a) Approx. A 3.5 kbp EcoRI / SalI insert from L. lactis or a functional fragment thereof, or

b) hybridizable with the above insert or a functional fragment thereof, or comprising a promoter region naturally operably linked to such a hybridizing sequence; or

c) an indicator peptide corresponding to (a) or (b), ··········································································· ·

- a degenerate version of 23 coding DNA sequences, or

(d) a derivative of a DNA molecule according to (a), (b) or (c), and / or

e) (I) a 2.5 Md plasmid of L. lactis LL712, or (II) a 5.2 Md plasmid of L. lactis LL712, or (III) a 2.5 Mdos or L. lactis LL712 , Contains the origin of replication of a plasmid belonging to the same incompatibility group as its 2 Md plasmids.

These DNA molecules are useful for making hybrid vectors of the invention or for searching for similar DNA or mRNA molecules in DNA or mRNA gene libraries.

Preparation of DNA molecules and MSP

The invention further relates to a process for the production of DNA molecules of the invention, i.e. hybrid vectors or DNA molecules as defined above. The procedure involves

A) culturing a host containing the DNA molecule of the invention and isolating the DNA molecule from the cultured host, or

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

Host cultures in conventional media may be performed which are negative or positive for the transformants. may contain, or may lack, certain compounds. Selection of transformants means the separation of hosts carrying the desired DNA molecule from hosts not carrying the desired DNA molecule based on the selection marker gene.

Any suitable host organism can be used for the process. Suitable hosts include bacteria such as Gram-negative bacteria such as E. coli or Gram-positive bacteria such as Bacillus species or lactic acid bacteria such as Lactococcus species, especially L. lactis, L. lactis diacety1actis or L. . cremoris.

Bacteria can be transformed by known methods, and transformants can also be identified by known methods such as resistance such as tetracycline resistance.

The hybrid vectors described above are particularly suitable in E. coli strains such as TG1, HB101, JM109, MH1 or the like, or in suitable Bacillus strains, or in suitable lactic acid bacteria such as Lactococcus strains such as L. lactis 0230, L. lactis diacetylactis, L. cremoris or the like, may be propagated. The hosts could be transformed and selected in known manner; the amplified plasmid DNA from bacteria is also conventional, e.g.

<img file="HUT57263A_D0009.tif" />

Doly (1979).

The DNA molecules of the invention may also be prepared by in vitro synthesis by known methods. This method is particularly suitable for the preparation of smaller DNA fragments, such as the prornoterenes of the MSP gene or similar genes, or particularly the coding DNA sequence of the signaling peptide.

The DNA molecules of the invention may also be obtained from lactic acid bacteria containing such molecules, in particular through their gene library or through their mRNAs.

In the following, the plasmid pUCRS has an approx. The production of a 3.5 kbp DNA molecule constituting an EcoRI / SalI insert from L. lactis is described in detail.

The gene library of a Lactococcus species such as L. lactis 0230 was used as starting material. The gene library is conventionally digested from L. lactis strain LM0230, C2 or LL712 strain by digestion with restriction enzymes (such as Sau3A or Mbol), and then in the appropriate vector (e.g. broadly).

The source of the gene library may be any other lactic acid bacterial strain producing MSP; other vectors may also be used to clone the DNA fragments.

In order to be able to select the invention from the gene library1,

26 DNA, a sample DNA that is capable of hybridizing to the desired sequence, such as the structural gene of MSP1, is required. It can also be a synthetic DNA fragment if the sequence of the MSP gene is, at least in part, known. Because the sequence of both the MSP protein and the MSP gene was unknown prior to the invention, we first had to purify the MSP, determine its N-terminal sequence, and generate hybridizable sample DNA.

MSP is classified as the most abundant gene product of Lactococcus species, such as L. lactis, in the culture medium, as determined by trichloroacetic acid precipitation of the medium, separation of precipitated proteins by SDS-polyacrylamide gel electrophoresis and staining with Coomassie Blue.

MSP can be prepared from any feedstock containing it, such as lactic acid bacteria such as Lactococcus trees such as L. lactis. Ά Purification can be accomplished by SDS-polyacrylamide gel electrophoresis followed by excision of the most protein-containing slices from the gel and dissolution of the MSP.

Purification of MSP by other means, such as precipitation with trichloroacetic acid, desalting, re-precipitation in the form of different salts, chromatography (affinity, ion exchange or gel permeation chromatography).

<img file="HUT57263A_D0010.tif" />

Electrophoretic methods (SDS-polyacrylamide gel, isoelectric focusing, electroelution or the like) or a combination of different methods can also be used.

In the development of the present invention, an approx. MSP of 56 kd apparent molecular weight was isolated in pure form from L. lactis LM0230 medium by precipitation with trichloroacetic acid, separation of the precipitated proteins by SDS-polyacrylamide gel electrophoresis1, excision of the gel containing the highest protein content, and electrophoresis of MSP. This method is suitable for the isolation of proteins similar to MSP but of other apparent molecular weights. The amino acid sequence of MSP was determined in part by direct sequencing and was derived in part from the DNA sequence; the result is shown in SEQ ID No. 1, where the secreted protein is from 28 to 466 amino acids.

Pure MSP protein is also within the scope of the invention.

Sequencing of the N-terminus of MSP was carried out in the usual manner and gave the following result:

<td>X -</td><td>X -</td><td>Asn</td><td>- Ser -</td><td>asp </td><td>- Ile</td><td>- Down</td><td>- Lys -</td><td>Gin - Asp</td>

<td>- Down -</td><td>Thr -</td><td>I le</td><td>- Ser -</td><td>X</td><td>- Down</td><td>- Gin</td><td>- Ser -</td><td>Alá - Lys</td>

<td>- Down -</td><td>Gin -</td><td>below</td><td>- Gin -</td><td>below </td><td>- Gin</td><td>- Val</td><td>- Asp -</td><td></td>

<td>The first</td><td>two</td><td>and the</td><td colspan="2">fifteenth</td><td colspan="2">that make a difference</td><td colspan="2">—In their place, X is 1</td>

- not defined.

• ··· • · · · · · · • · · · ··· · ······ · ·· · • ·· · ·«·· · · · ·

- 28 Based on the amino acid sequence between positions 5 to 13, the following oligonucleotide mixture was synthesized:

GANI ATN₂ GCI AAN₃ CAN₃ GANI GCI AC

In the above sequence, Ni = T or C, N₂ = C or A, N₃ = A or G (A = adenine, T = thymine, C = cytosine, G = guanine, I = inosine). The oligonucleotide mixture was labeled with radioactivity in the usual manner and used as sample DNA for the study of the Lactococcus species library, in particular L. lactis LM0230 gene library.

Sample DNAs encoding the amino acid sequence of MSP and having a length of at least 14 bp are also capable of selecting DNA molecules carrying genes or fragments similar to the structural gene of MSP and mRNA molecules encoding pre-MSP.

The sample DNAs were labeled with radioactivity at their 5 'end; the marking in a well known manner,³²P-ATP and T4 kinase were performed. Host microorganisms or phages containing the nucleic acids of the invention as inserts were identified by membrane replication of their gene library by hybridization with a radioactive sample.

Hybridization conditions were standard; the severity of the conditions was influenced by changing the temperature.

b · »» ♦ »• · · • · 4 •« «·· ··

The residual portion of the DNA library hybridizes with the oligonucleotide mixture of oligonucleotides, partially - in a known, conventional manner; the defined sequence comprises almost the entire MSP gene. The known sequence is shown in SEQ ID No. 1.

The 3.5 kbp L. lactis EcoRI / SalI insert can be isolated by digesting the positive clones of the gene library with EcoRI and SalI nucleases and then purifying the fragments by known methods, such as agar steam electrophoresis1. The fragments isolated in this manner can also be linked to the appropriate vectors by known methods: from M13mpl8 to M13mpl8RS or from pUC18 to p'JC18RS P1 plasmids.

Similarly, any other derivatives, fragments, or variants of any other L. lactis insert carrying the MSP sequence, such as the 3.5 kbp EcoRI / SalI insert, or DNA molecules capable of hybridizing with the insert, or a DNA molecule capable of hybridizing to the insert, may be naturally isolated. related promoter region from gene libraries of lactic acid bacteria.

Fragments of the DNA molecules corresponding to (a) to (c) are conventionally used, for example, as an exon or endonucleotide suitable for insertion, such as exonuclease III. Bal31 or S1- or restriction endonucleases1 -pl. digestion of Sau3A, Hindi II, and the like, and the fragments generated. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · R · ··

- 30, but can also be obtained by in vitro synthesis by conventional methods.

Mutants of the DNA molecules corresponding to (a) to (c), such as inversion, deletion, insertion or point mutants, are known methods such as in vivo or in vitro directed mutagenesis (Zoller and Smith (1983); Botstein and Shortle (1985); Norris et al. (1983)], can be prepared using mutagenic oligonucleotide primers or by enzymatic excision of the DNA between two restriction sites and ligation of the ends. The preparation is preferably carried out in dilute solutions.

The preparation of the recombinant DNA molecules of the invention, including the DNA sequence which acts as the origin of replication, will now be described in detail.

Plasmids of L. lactis LL712 were isolated following the method of Bimbóim and Doly (1979) (modifications exemplified) and then separated by conventional means using chromatography techniques such as agarose gel chromatography (Maniatis et al. (1982)). The 2.5 Md and 5.2 Md plasmids were isolated and fragmented in the usual manner.

The resulting mixture of fragments was coupled to a suitable vector, also known in the art (Maniatis et al. (1982)), and transformed with a mixture of ligands into a suitable intermediate host strain. '• *

<img file="HUT57263A_D0011.tif" />

- 31 Ά of a suitable vector also carries a marker gene which makes it suitable for selection in lactic acid bacterial cells. Such markers may be resistance genes such as erythromycin resistance. The origin of replication of the corresponding vector does not work in lactic acid bacteria, but it does work in intermediate host bacteria. The marker vector of the appropriate vector is such that it also provides for selection of the intermediate host, i.e. it functions in the same manner in the intermediate host and in the lactic acid bacterial urn. Suitable intermediate hosts include various strains of E. coli, such as E. coli TG1; a suitable vector is an E. coli vector, such as a variant of erythromycin resistance pUC18, such as pUC383, the construction of which will be described in the Examples.

Intermediate host cells transformed with vectors carrying the 2.5 Md and 5.2 Md ρ1 plasmids fragments of L. lactis can be selected by known methods, depending on the type of host cell and vector used. Selection markers may be, for example, resistance genes or genes for well-detectable enzymes, such as the 1acZ gene of β-galactosidase, provided that the E. coli strain used by the host is detective for this gene. Insertion of the DNA fragment may damage the vector marker gene; if the marker gene is 1 acZ. so host cells that have transduced the 1acZ gene in their transforming vector will not be able to produce X-Gal ι

To convert to C · · · 4 · · 4 · · · · · · 9 ······································································································· colonies of cells transformed with vectors remain white instead of blue staining for lacZ.

Vectors carrying inserts derived from L. lactis 2.5 Md and 5.2 Md plasmids were tested for their ability to replicate in a plasmid-free L. lactis strain, such as L. lactis 0230. For this purpose, the plasmids were isolated from the intermediate host in a conventional manner. L. lactis 0230 cells were transformed with Powe11 et al. (1988). Amplifiable plasmids are those whose DNA insert also contains origins of replication of L. 1actis-p1 plasmids. The DNA molecules containing the origin of replication may be isolated from the amplified vector, fragmented, mutated, etc., and used to construct recombinant DNA molecules of the invention, i.e., cloning or expression vectors capable of replication in lactic acid bacteria. Because the replication origins of the 2.5 Md and 5.2 Md plasmids of L. lactis originate in bacteria other than lactic acid bacteria, such as Bacillus species or E. Vectors carrying such DNA fragments can also be used as a commuting vector.

Fragments of the replication origin of the 2.5 Md or 5.2 Md plasmids can be identified and isolated by fragmentation of the complete p 1 asmid-ready 1 ethid of L. lactis LL712.

- from a mixture of 33 fragments obtained by hybridizing the cloned fragments with the complete plasmids of L. lactis LL712 separated on an agarose gel, or by comparing the restriction patterns of the cloned fragments with the original plasmids.

Recombinant DNA molecules of the invention which are L. lactis 2.5 Md or 5.2 Md ρ1 plasmids, or plasmids of the same incompatibility group, can be obtained, for example, by opening the plasmid with an appropriate restriction endonuclease and ligating it with, for example, a fragment comprising a homologous or heterologous structural gene, a promoter region, a vector sequence, a linker region or the like. Restriction endonucleases having only one recognition site, i.e., a cleavage site, are suitable for the method outlined.

A recombinant DNA molecule containing the origin of replication of one of the 2.5 Md or 5.2 Md plasmids can be prepared, for example, by isolation of the L. lactis LL712 plasmid pool in a known manner, identified by agarose gel 2, The 5 Md and 5.2 Md plasmids were fragmented with appropriate restriction enzymes, the fragments containing the origin of replication were isolated, and the fragments or mixtures were ligated with one of the replication {

A DNA molecule containing no 34 sites, such as a detective cloning vector, and selecting DNA molecules capable of replication in at least L. lactis and E. coli.

Vectors containing origin of replication of plasmids belonging to the same incompatibility group as 2.5 Md or 5.2 Md plasmids are recognized as being unable to survive with their 2.5 Md or 5.2 Md plasmids. in the same host cell.

The invention encompasses the use of a DNA molecule or a recombinant DNA molecule of the invention for the production of a hybrid vector expressing a structural gene; examples of such structural genes have already been given. Hybrid vectors can be prepared by conventional techniques using enzymes such as restriction enzymes, DNA polymerases, DNA ligases, and the like.

The construction of hybrid vectors expressing structural genes in lactic acid bacteria, especially L. lactis, or Bacillus species, particularly B. thuringiensis, is described in detail in Examples 4 and 5.

Transformed hosts and their establishment

Also included within the scope of the invention are bacteria transformed with the hybrid vectors of the invention.

···· ♦ · · · ··· » • ···· · · · · · ··· · ·«·· ·· ··

The transformed bacteria of the present invention are capable of cloning, amplifying and / or producing hybrid vectors carrying the DNA molecules of (a) to (e). In such hosts, the hybrid vectors replicate and are not lost under selection pressure during bacterial population growth. The choice of host may depend on the origin of replication of the vector. In the case where the hybrid vector carries a DNA sequence including an origin of replication according to (e), an appropriate host can be, for example, any strain of E. coli, Bacillus or Lactococcus species, provided they do not carry a a plasmid whose origin of replication is in the same incompatibility group as the hybrid vector.

When the hybrid vector contains the homologous or heterologous structural gene, together with the DNA sequence encoding the MSP-tagging peptide, in an appropriate reading frame, the transformed host of the invention is an organism suitable for secretory production of the homologous or heterologous protein, e.g. -tribe.

Examples of transformed host organisms of the present invention include pUCRS, pSC12, pSC12deltaP, pSC12deltaN, pSC122de1taNP, pSC18, pSC18deltaN, pSC18deltaP, pVAHIR, PVAHIR-I, PSC12HIR, plasmid TSC12H1R1 or pSC12H1R1 or plasmid pSC12H1R1, C600 or · · »» »• • • • 4 4 4 4 4 4 4 4 4 4 4 4

36 ΗΒ101), L. cremoris, ρ1 plasmid-free L. lactis (e.g. LM0230), or Bacillus species (e.g. B. thuringiensis). L. lactis transformed with plasmid pUCRS, L. lactis transformed with pUCRS, pSC12HIRTerm, pSC12 or pSC18, or B. thuringiensis transformed with pVAHIR or pVAHIR-1.

Also included within the scope of the invention is a process for the preparation of such transformants, which comprises treating the host organism with recombinant DNA molecules of the invention, in particular hybrid vectors of the invention, preferably a hybrid vector comprising a selectable marker; as well as the selection of transformants.

A process for preparing polypeptides

The present invention further provides a method for producing polypeptides comprising the step of linking a gene having a homologous or heterologous structure to a suitable reading frame by a DNA sequence encoded by the MSP-tagging peptide; a suitable host organism - a Gram-positive bacterium such as Lactococcus such as L. lactis LM0230 or a Bacillus species such as B. thuringiensis - transformed with a hybrid vector carrying the above gene structure; and that the polypeptide encoded by the above gene • · · ·

- 37 are secreted from the host cell. If desired, the polypeptide is isolated from the medium by conventional means. A preferred embodiment of the method is the use of a bacterium which does not produce a protease that would degrade the expressed polypeptide in the medium.

Such a preferred embodiment of the invention is the production of proteins, such as desulfohirudin, which are separated by L. lactis LM0230 cells transformed with a hybrid vector carrying this gene (e.g., pSC12HIR, pSC12HIR-1 or especially pSC12HIRTerm). The active desulfo-hirudin in concentrated form can be obtained from the supernatant of a stationary phase culture of L. lactis LM0230 cells transformed with PSC12HIR or PSC12HIR-1. L. transformed with plasmid pSC12HIRTerm. lactis LM0230 cell supernatant has an increased yield of desulphohirudin. Surprisingly, the level of heterologous gene products, such as desulf o -hirudin, is not prolonged, e.g., overnight incubation, indicating that there is no protein-degrading enzyme in the supernatant of the stationary phase culture that could degrade the heterologous gene product.

In another embodiment of the method of the invention, the cells are harvested (e.g., by centrifugation or filtration) from a slurry or stationary phase culture and resuspended in fresh medium or from 1% to 20% of their original volume in a suitable buffer. THE • · · · %

<img file="HUT57263A_D0012.tif" />

Incubation of 38 resuspended cells enhances the yield of heterologous gene product: for example, desulfochohirudin production of L. lactis LM0230 cells transformed with plasmid pSC12HIR is increased by centrifugation of the cells from the stationary phase and incubation in 1/10 volumes of fresh medium. C temperature.

Another embodiment of the method of the invention is the production of MSP secreted by Gram-positive bacteria transformed with one of the hybrid vectors of the invention, such as the pUCRS plasmid.

Also within the scope of the invention is the production of desulfohirudin in B. thuringiensis transformed with plasmids pVAHIR or pVAHIR-1, preferably strain HD1cryB (DSM 4574).

Brief Description of the Drawings

First figure. Physical map of plasmid pUC838.

The positions on the map are approximate and are expressed in kbp. The pUC18 moiety is 0 to 2.7 kbp, the pVA838 moiety, which carries erythromycin resistance, the 2.7 to

4.4 kbp sequence. The restriction sites enclosed in parentheses were deleted during the preparation of the recombinant molecule. Abbreviations: amp^r = ampici11 resistance gene; ery^r = erythromycin resistance gene; ori_plac: = origin of replication

<img file="HUT57263A_D0013.tif" />

- 39 from plasmid PUC18.

Second figure. Physical map of plasmid pSC12.

The positions on the map are approximate and are expressed in kbp. The pUC18 DNA is 0-2.7 kbp, the pVA838 fragment - which carries vascular thromycin resistance -

The 2.7 to 4.4 kbp insert from the 2.5 Md plasmid of L. lactis has a sequence of 4.4 to 8.0 / 0 kbp. The restriction sites enclosed in parentheses were deleted during the preparation of the recombinant molecule. Abbreviations: PL1 = pUC polylinker region from EcoR1 to (Smal); PL2 = pUC polylinker region from (Smal) to Sphl; amp * - = ampicillin resistance gene; ery^r = erythromycin resistance gene; oripuc = origin of replication from pUC18; orin = origin of replication from Lactococcus 2.5 Md (deduced from deletion analysis).

Third figure. Physical map of plasmid pSC18.

The positions on the map are approximate and are expressed in kbp. The pUC18 DNA is 0-2.7 kbp, the pVA838 fragment which carries erythromycin resistance.

2.7 to 4.4 kbp, the insert from the 5.2 Md plasmid of L. lactis has a sequence of 4.4 to 8.0 / 0 kbp. The restriction sites enclosed in parentheses were deleted during the preparation of the recombinant molecule. Abbreviations: PL1 = the polyUC linker region of pUC from EcoR1 to (Smal); PL2 = a pUC polylinker ί

«

<img file="HUT57263A_D0014.tif" />

- 40 areas from (BamHI) to HindIII; amp^r = ampici11 resistance gene; ery^r = genes for resistance to chorythromycin; oripuc = origin of replication from pUC18; orii₂ = origin of replication from Lactococcus 5.2 Md (deduced from deletion analysis).

Abbreviations dNTP deoxynucleotide triphosphate

HPLC High Performance Liquid Chromatography

IPTG isopropyl-O-D-thiogalactopyranoside kbp kilobase pair kd per kilodalt

LB Luria General Nutrient Fluid (Gibco / BRL)

Soluble CD23, a 25 k IgE binding factor of sd23, was md

SLPI is a secretory leukoproteinase inhibitor

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

• · · · ··· · ···· ·« · ♦

- 41 Materials and Methods

Bacterial strains

L. lactis LM0230: a C1-free derivative of L. lactis C2 (Efstathiou and McKay (1977)); deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (see later).

L. lactis C2 (NCDO 2031) and L. lactis LL712: the two strains (the latter obtained plasmids 2.5 Md and 5.2 Md) are very similar or identical (Davis et al., 1981). the former is available, for example, from the National Collection of Diary Organisms (UK), the latter has been deposited with DSM.

E. coli TG1: genotype K12, delta (lac-pro), supE, thi, hsdD5 / F'traD36, proA⁺B⁺, lacl^q, lacZdeltaM15; the strain is sold by Amersham, with a detailed description (Oligonuc1eotide-directed in vitro mutagenesis system).

General methods

Transformation

E. coli was transformed using the calcium chloride method described by Maniatis et al. (19Θ2).

Ά Lactococcus strains by electroporation, a

BioRad Gene Pulser^R was transformed using the procedure described by the company for L. lactis strain LM0230.

• · ·· ·· ···· • · · · · · · • · · · · ♦ · · • ···· · · · · · • · · · ···· ·· ··

Preparation of plasmids

Plasmid DNA was obtained from E. coli by the method of Bimbóim and Doly (1979).

The same method was used for isolation from Lactococcus1 with the following modifications:

(a) Cultures incubated in M-17G medium (Terzaghi and Sadine (1975)) at 30 ° C overnight: diluted 10% in fresh medium and incubated for another two hours;

b) In addition to the lysozyme, the cells were also treated with mutanolizine (50 mg / L) at 37 ° C for 10-20 minutes.

The L. lactis LL712 plasmid set was similarly isolated and the plasmids were further purified by cesium chloride-ethidium bromide gradient centrifugation (Clewell and Helsinki, 1969). The three smaller [1.8 Md, 2.5 Md and 5.2 Md; Gasson (1983)] was enriched in the purified fractions, whereas the two larger plasmids (9 Md and 33 Md) were only trace.

First example. Preparation of commuting vectors for lactic acid bacteria and E.

coli

1.1 Construction of plasmid pUC838

A pVA838 [ATCC 37160; Macrina et al. (1982)] a · · · · · · · · · · · ···

43 Lactococcus p1 plasmid containing a constitutively expressed erythromycin resistance gene on a 1.7 kbp Aval / HindIII fragment. About 10 µg of pVA838 DNA was digested with Aval and HindIII. The ends of the fragments were quenched with KIenow enzyme1 in the presence of all four dNTPs, then separated on an agar gel and the 1.7 kbp Aval / HindIII fragment was recovered from the gel by electroelution.

Approximately 200 ng of the above fragment was ligated to 100 ng of plasmid pUC18 'opened with SmaI endonuclease, following the method of Rusche et al. (1985), and E. coli TG1 bacteria were transformed with the ligation mixture. On the LB agar plate containing 100 mg / L ampicillin, X-Gal and IPTG, 1 overgrown white colonies were highlighted and the corresponding

It was identified by growth on LB agar plate containing 1 mg / l erythromycin and by analysis of plasmid DNA. The plasmid thus created was designated pUC838; restriction sites and major features thereof are shown in Figure 1.

1.2. Construction of plasmid pSC12 The plasmid kit isolated from L. lactis LL712 was separated on a preparative agarose gel; a second DNA band containing the 2.5 Md plasmid was excised and the plasmid was subtracted by electroelution. After digestion with Sphl endonuclease and agarose gel separation, 4 4 · · · · · · · · · · · · · · · · · · · · · · · · ·

44 single 3.5 kbp fragments were obtained, indicating that the 2.5 Md plasmid had only one or a few but very close SphI sites.

The single SphI site of plasmid PÜC838 was opened and treated with calf intestinal phosphatase, followed by approx. 50 ng of treated vector and 150 ng of 3.5 kbp SphI fragment were ligated with T4 ligase. E. coli TG1 cells were transformed with the ác ligation mixture, and the bacteria were first incubated at 37 ° C in 1 ml of LB medium for 90 minutes, then transferred to 200 ml of LB medium containing 100 mg / L erythromycin and further incubated overnight. The cells cultured in this way were subtracted from the ρ1 plasmid DNA and extracted approximately 1 µg from it. Two µg of L. lactis LM0230 cells were transformed and transformants were selected on M-17G medium containing 5 mg / L erythromycin at 30 ° C.

The ρ1 plasmid DNA was isolated from several clones of transformed L. lactis LM0230. All transformants tested contained an 8.0 kbp plasmid, called pSC12, from which the 3.5 kbp SphI insert was recoverable. The physical map of pSC12 is shown in Figure 2.

Plasmid pSC12 was shaken several times over L.

lactis and E. coli without a noticeable change in the restriction pattern.

• · · ·

1.3. Construction of plasmid pSC1S

The plasmid of L. lactis LL712 was partially digested with Sau3AI endonuclease by treatment of 500 ng portions of DNA with different amounts of enzyme. Samples were identified in an agar ten gel and the partially digested DNA was ligated to pUC838 plasmid opened with BamHI endonuclease and treated with calf intestinal phosphatase.

Transformation of E. coli TG1 followed by L. lactis LM0230 with the resulting plasmid was performed according to section 1.2. The 8.8 kbp plasmid derived from the transformants, called pSC18, was also assayed. Movement of the plasmid between L. lactis and E. coli did not alter the restriction pattern, demonstrating the stability of the plasmid in both cell types. The physical map of pSC18 is shown in Figure 3.

Second example. Identification of the Lactococcus origin of replication origin

2.1. Deletion analysis of plasmids pSC12 and pSC18

Deletion analysis was performed on plasminides pSC12 and pSC18 to identify the origin of Lactococcus origin of replication.

DNA sections between the appropriate restriction sites were excised with the appropriate restriction endonucleases.

« ·«·

<img file="HUT57263A_D0015.tif" />

and then the plasmid is recirculated to T4-1 ligase 1. The approximate location of the restriction sites is shown in Figures 2 and 3 and Table 1.

The pUC18 portion of plasmids pSC12 and pSC18 was excised between the two PvuII sites - containing the col E1 origin of replication - to give plasmids pSC12deltaP and pSC18deltaP respectively.

Removal of the smaller NdeI fragment from pSC12 and pSC18 gave plasmids pSC12deltaN and pSC18deltaN.

Removal of the EcoRV fragment from pSC12 gave plasmid pSC12deltaR.

The plasmid pSC12deltaNP was excised from the PvuII restriction site of the pUC portion of pSC12 to the NdeI site of the Lactococcus-derived portion; in this case, the ends were blunted with Kenow enzyme1.

The DNA fragment between the HindIII restriction sites was excised to obtain plasmid pSC18deltaH from pSC18.

All mutant plasmids were first isolated from erythromycin resistant transformants of E. coli TG1 and then tested for their ability to transform L. lactis LM0230; erythromycin resistance was used as a marker. In case of failure, we assumed that the plasmid was absent or damaged by deletion of the Lactococcus

<img file="HUT57263A_D0016.tif" />

origin of replication. Plasmids were isolated from erythromycin resistant transformants and their structural integrity was confirmed by restriction analysis.

In both your 2.5 Md and 5.2 Md plasmids, the area required for growth in the 1-lactococci was identified. The fact that de 1taP derivatives are capable of replication in E. coli also demonstrates that Lactococcus origin of replication is also functional in Gram-negative E. coli.

The results of the experiment are shown in Table 1.

First spreadsheet. Result of deletion analysis of plasmids pSC12 and pSC18

Plasmid Cut Sequence Replication site¹ length² E.coli L.lactis

<td colspan="3">pSC12</td><td> +</td><td> +</td>

<td>pSC12deltaP</td><td>PvuII (0, l) -PvuII (2.5)</td><td> 2,4</td><td> +</td><td> +</td>

<td>pSC12deltaN</td><td>Nde1 (0.2) -Nde1 (6.2)</td><td> 2,0</td><td> +</td><td> +</td>

<td>pSC12deltaR</td><td>EcoRV (5.0) -EcoRV (7.5)</td><td> 2,5</td><td> +</td><td> -</td>

<td>pSC12deltaNP</td><td>Nde1 (6,2) -Nde1 (2,5)</td><td> 4,3</td><td>NV³</td><td> +</td>

<td>pSC18</td><td></td><td></td><td> +</td><td> +</td>

<td>pSC18deltaP</td><td>PvuII (0.1) -PvuII (2.5)</td><td> 2,4</td><td> +</td><td> +</td>

<td>pSC18deltaN</td><td>Ndel (0.2) -Ndel (7.5)</td><td> 1,2</td><td> +</td><td> +</td>

<td>pSC18deltaH</td><td>Hindi 11 (0) -PriceI11 (6.5)</td><td> 2,0</td><td> +</td><td> -</td>

• · «· ·« ·»«* • * · · 9 » · • · · ···· « • ·*·· · » ·· · • · · · »·«· ·· ··

- 48 ^XThe excised sequences were between two restriction sites, calculated from the HindIII restriction site (selected as 0) of the pUC838-derived portion of the plasmids and indicated in parentheses. The positions shown are the same as those shown in Figures 2 and 3.

²The length of the excised sequence in kbp.

³We didn't investigate.

2.2. Source of Lactococcus-derived Jr'ep 1 seedlings

Plasmid pSC12 originates replication from L. lactis LL712

Derived from its 2.5 Md plasmid; this is clearly evident from the way in which the DNA fragment carrying the origin of replication is isolated.

It is apparent from the restriction pattern of plasmid pSC18 that the source of the replication origin is plasmid 5.2 Md.

2.3. Replication of plasmids pSC12 and pSC10 with other Lactococcus

mortalities,

Different strains of L. lactis, L. lactis diacetylactis and L. cremoris were transformed with both plasmids; plasmids were stable in populations below selection selectivity.

Λ 1 ····

Third example. Ά Cloning of the L. lactis LM0230 MSP gene

3.1. Isolation of L. lactis LM0230 MSP

1.5 L of L. lactis LM0230 culture medium (M-17G medium, 30 ° C, overnight) was centrifuged (Sorvall GS-3 rotor, 7000 rpm, 20 min, 4 ° C), and the supernatant was collected and an equal volume of ice-cold 10% trichloroacetic acid was added. The mixture was allowed to stand for 30 minutes at room temperature and the precipitated proteins were recovered by centrifugation (Sorvall GS-3 rotor, 8000 rpm, 30 min, 4 ° C). The precipitate was redissolved in 3 mL of SDS sample buffer (Laemmli (1970)), neutralized with a small amount of 4 M sodium hydroxide, and dialyzed with 4 L of 25 mM Tris-HCl buffer, pH 6.8, 0.02% for 4 hours. SDS). The volume of the solution after dialysis is approx. It was 4 ml, which was adjusted to 1 6 ml for 3x SDS sample buffer.

Proteins were separated in a volume of 2-2 ml on an 8% SDS-polyacrylamide gel (Laemmli (1970)) using a BioRad Protean cell preparative electrophoresis apparatus. The thin strips of the gel were stained with Coomassie Blue and then colorless with an aqueous solution of 10% methanol and 10% acetic acid. These bands were used to label and cut out the largest protein band from the gel; the protein had an apparent molecular weight of 56 kd.

The MSP was eluted from the gel by electroelution (150 V for 2 hours).

·· ····

- 50 Biotraps) was extracted with 20 mM ammonium acetate buffer (0.01% SDS). The extracted protein solution was dialyzed against 20 mM ammonium acetate buffer (0.005% SDS) for 48 hours and then a sample was run on an 8% SDS-polyacrylamide gel to give a crude protein band with an apparent molecular weight of 56 kd.

3.2. Determination of the N-terminal sequence of MSP

The N-terminal amino acid sequence of MSP was routinely determined on a gas phase sequencer1 (Applied Biosystems Inc., Model 470A) followed by HPLC identification of the phenylthiohydantoin derivatives of the cleaved amino acids. The first 28 amino acids are:

<td>X -</td><td>X -</td><td>Asn</td><td>- Ser -</td><td>asp</td><td>- Ile</td><td>- Down</td><td>- Lys</td><td>- Gin - Asp -</td>

<td>- Down -</td><td>Thr -</td><td>Ile</td><td>- Ser -</td><td>X </td><td>- Down</td><td>- Gin</td><td>- Ser</td><td>-Alá - Lys -</td>

<td>- Down -</td><td>Gin -</td><td>below</td><td>- Gin -</td><td>below</td><td>- Gin</td><td>- Val</td><td>- Asp</td><td> -</td>

<td>The first</td><td>two</td><td>and the</td><td colspan="2">fifteenth</td><td colspan="2">amino acids</td><td colspan="2">—The X is in place—</td>

- not defined.

3.3. Synthesis of sample oligonucleotide mixture

N-terminal amino acids 5 to 13 have the following sequence:

- Asp - Ile - Ala - Lys - Gin - Asp - Ala - Thr - Ile, an oligonucleotide mixture was designed and prepared for DNA library screening. It consists of 23 bases

<img file="HUT57263A_D0017.tif" />

·· ···· • ♦ ·· ·· * · » · • · · · · • ···· · 4 * · · ··· · ···· ·» ··

- Inosine was inserted at two points of 51 oligonucleotides where all four dNTPs would have had to be used due to code degeneracy to reduce the variation potential of the mixture. The resulting oligonucleotide mixture has the sequence:

GANI ATN₂ GCI AAN₃ CAN₃ GANi GCI AC

In the above sequence, Ni = T or C, N₂ = C or A, N₃ = A or G (A = adenine, T = thymine, C = cytosine, G = guanine, I = inosine).

3.4. Preparation of the L. lactis LM0230 gene library

L. lactis LM0230 chromosomal DNA was isolated in a manner similar to plasmids except that after treatment with lysozyme and mutanolizine, the cells were incubated for 2 hours with 100 mg / L proteinase K, 20 mmol EDTA and 0. Tris-HCl buffer (10 mmol, pH 8.0) containing 5% SDS at 56 ° C. The DNA was extracted with phenol: chloroform = 1: 1 and purified by centrifugation in a gradient of cesium chloride.

The resulting chromosomal DNA was partially digested with Sau3AI endonuclease and then fractionated by size in a sucrose gradient according to Maniatis; fragments of 10-20 kbp were collected.

LambdaEMBL3 phage DNA was cleaved with BamHI and EcoRI endonucleases followed by phenol extraction and ethanol.

4 4 · ···4 • ···· ·44··

444 4 4444 444«

- We cleaned it with 52 precipitations. The resulting DNA and 10-20 kbp fragments were ligated in the presence of the cobalt (III) chloride hexamine binding promoter (Rusche et al. (1985)) and the ligation mixture was transformed in vitro into Stratagene Inc. Gigapack Plus^Rsystem.

The gene library was propagated in E. coli strain Q359 (Kahn et al. (1980)) and approximately 600,000 recombinant phages were obtained.

3.5. Finding MSP coding sequences in the gene library

Cultures of recombinant lambdaEMBL3 phages (about 15,000 plaques on a 15 cm plate) were prepared in Petricese and transferred to PlaqueScreen^R (NEN) membranes. The membranes were hybridized with the sample oligonucleotide mixture, which was T4-kinase and³²P-ATP was labeled with radioactivity according to Maniatis et al. (1982).

Positive plaques were identified and tested a second time with a low plaque density of 1t.

DNA from the positive phages was isolated, digested with restriction enzymes, and analyzed by Southern blot with the mixed oligonucleotide sample. The sample hybridized with the 3.5 kbp EcoR-I / SalI fragment of the positive phage L. -lactis, further studies revealed

<img file="HUT57263A_D0018.tif" />

53, however, that a 2.1 kbp HindIII fragment carries the hybridizing sequence.

The relative position of the restriction sites of the 3.5 kbp Eco RI / Sal I fragment relative to the Eco RI end was examined by digesting the fragment with appropriate enzymes or enzyme mixtures and then by agarose gel electrophoresis. The results showed that the first HindIII restriction site was about 1. 0.5 kbp, the second approx. 2.6 kbp and the third approx. 3.15 kbp from EcoRI end.

3.6. Construction of plasmid pUCRS

Positive phage DNA was digested with EcoRI and SalI endonucleases, the fragments were separated on 0.6% agarose gel, the 3.5 kbp EcoRI / SalI fragment was excised and isolated by electroelution. Three times the molar amount of the fragment was ligated to pUC19 Ρ1 opened with EcoRI and SalI endonucleases and then treated with alkaline phosphatase.

E. coli TG1 cells were transformed with the ligation mixture and the transformants were selected on an agar plate containing 100 mg / L ampicillin. Plasmid DNA was prepared from the transformants and confirmed by restriction analysis. The plasmid thus obtained is pUCRS.

The E. coli strain TG1 carrying plasmid pUCRS was deposited: - 54

ij was placed in the Deutsche Sammlung von Mikroorganismen und Zell | kulturen at.

I

I i

3.7. Sequencing of the MSP gene

Sequencing for the Chain Termination Method1 (Sanger et al. 1977), USBC Sequenase^R system.

The 3.5 kbp EcoRI / SmaI fragment was prepared as described above and subcloned into your M13mpl8 plasmid: the resulting plasmid is M13mpl8RS. The 2.1 kbp Hindi II fragment obtained from the A-3.5 kbp EcoRI / SmaI fragment was subcloned into M13mpl9 plasmid to give M13mpl9H. Plasmid M13mp18RS is exonuclease III and Si (Henikoff (1984); Yanish-Perron et al. (1985)]; this creates a series of unidirectional deletions from the Sallj end of the insert, which are approx. 2 up to kbp in the inertia. These deletions were sequenced starting from the universal mpl8 primer, thus obtaining the sequence of one strand. Given the sequence thus obtained, a number of synthetic ι oligonucleotides were generated which served as primers for sequencing the second strand. Additional sequence data were obtained by examining exoIII-S1 deletions from plasmid M13mp19H. The resulting 1920 bp DNA sequence is shown in SEQ ID NO. 1 This contains the promoter region of the MSP gene, the MSP signaling peptide

<img file="HUT57263A_D0019.tif" />

coding sequence and the structural gene of the mature MSP.

Sequence analysis of about 2 kbp resulted in an open reading frame encoding a 461 amino acid protein. Comparing the amino acid sequence deduced from the base sequence with the amino acid sequence obtained by sequencing the N-terminus of MSP, the mature MSP is composed of 434 amino acids preceded by the 27 amino acids in the reading frame. The peptide formed from the latter has the structure of the peptide. a typical structure of the tagging peptides: the charged amino acids are located at the N-terminus and most of the other amino acids are hydrophobic and the structure of the cleavage point is consistent with the structure of the known tagging peptides (Van Heinje (1983)).

4th example. Production of recombinant desulfo-hirudin in Lactococcus

In 1act is

4.1. Construction of a plasmid encoding desulphohirudin

Plasmid M13mp19H carries the 2.1 kbp HindIII fragment which encodes the N-terminal half of the MSP with the tag peptide and also contains a DNA fragment of about 1.5 kbp. The plasmid was cut at a single Scal restriction site 129 bp downstream of the C-terminus of the tagging peptide. A blunt-ended 211 bp DNA fragment was isolated from plasmid pML310 (EP-A 168342;

<img file="HUT57263A_D0020.tif" />

this carries the genetic information for desulfo-hirudin) and was then inserted into the opened Scal restriction site of M13mpl9H. The reading frame was repaired by deleting the DNA coding sequence for the MSP tag peptide and the structural gene for desulfohirudin, thereby removing the last amino acid of the tag peptide. (Ala) and the codon for the first amino acid (Val) of desulfo-hirudin were aligned. Removal was achieved by in vitro mutagenesis with the oligonucleotide using the Amersham system; the 29 bp oligonucleotide used for this purpose contained the last 14 base pairs of the coding peptide coding sequence and the first 15 bp of the desufluoro-hirudin coding sequence. The complete DNA sequence obtained by fusion is shown in SEQ ID No. 2.

The HindIII fragment carrying the fusion gene was excised and inserted into a single HindIII restriction site of plasmid pSC12, and both bidirectional inserts were isolated and the plasmids carrying them were named pSC12HIR and pSC12HIR-1.

An additional plasmid (pSC12HIRTerm) was constructed in which the transcription terminator of the trpA gene of E. coli (Pharmacia) was inserted. The terminator was located at a single HpaI restriction site on plasmid pSC12HIR, about a half of the desulphohirudin gene. 125 bp downwardly inserted.

<img file="HUT57263A_D0021.tif" />

4.2. Production of desulphohirudin by L. lactis

With the fusion plasmids encoding desulphohirudin, L. lactis

LM0230 cells were transformed. The transformants were cultured in M-17G medium supplemented with 2% glucose and containing 5 mg / l erythromycin at 30 ° C. When the cultures reached the stationary phase, 1.5 mL miniatures were removed, centrifuged in an Eppendorf tube, and the supernatant frozen at -70 ° C, and the culture was further incubated overnight and repeated.

The desulfo-hirudin content of the samples was determined on the basis of biological activity.

The presence of desulfo-hirudin can be demonstrated by its anti-thrombotic activity. The test procedure is as follows;

0.2 M Tris-HCl buffer (pH 7.5, 0.1 M sodium chloride, 0.01% bovine serum albumin) was used to dilute the samples and reagents. Thrombin was derived from human plasma (Protogen AG, Laufel fingen. No. 80-13-1102) and the chromogenic thrombin substrate was Chromozyme TH (Boehringer, Mannheim, No. 206849). The p-nitroaniline released from Chromozyme TH was measured with a Dynatech MR600 plate reader photometer. All measurements were made in microtiter plates (Nunc, MicroWell plates). 50 µl of buffer, 50 µl of sample and µl of thrombin solution were added to each well and the reaction was started by adding 150 µl of substrate solution (330 µg / ml) and

• * • • «« «« «« «« «« «

The plates were incubated for 2 hours at 37 ° C. The concentration of the thrombus was adjusted so that the extinction of the control without the inhibitor at 405 nm was 0.8 ± 0.2.

Both the substrate and the thrombin solution were stored frozen at -20 ° C and thawed just before use. The calibration curve r [Tyr⁶³] solutions of desulfo-hirudin at known concentrations (40, 20, 10, 5, 2.5 and 1.25 ng / ml).

I {L. transformed with plasmids pSC12HIR or pSC12HIR-1.

in the supernatants of steady-state cultures of lactis LM0230, deszu1-hirudin activity was the same, whereas about 50% was higher in the culture of cells transformed with pSC12HIRTerm. As a control, with your pSC12 plasmid hirudins were not controversial in the culture supernatant of transformed cells.

The supernatant desulfo-hirudin level was not reduced when the cultures in the stationary phase were further incubated for 16 hours.

í

This indicates that the culture of L. lactis LM0230 is supernatant soy has no protein-degrading activity.

In one experiment, cells were harvested from the culture by centrifugation, the suspensions in 1/10 volumes of fresh medium, and incubated for 30 minutes at 30 ° C; ten. Measurements showed that the post-culture supernatant was six times higher than the previously measured desulphohirudin level,

<img file="HUT57263A_D0022.tif" />

• · ·· ·· ···· • · · · · · · • · · ♦ · · · · • ·«· · · · ·· · ··· · ···· ·« · ·

59 means that concentration of cells in the expression phase is a suitable method for increasing the concentration of the secreted product in the supernatant.

5th example, Production of recombinant desulfo-hirudin in Bacillus thuringiensis 1

5.1. Ά Construction of plasmid pVAHIR

Plasmid pVA838 (Macrina et al. (1982)) was digested with HindIII endonuclease and contained approximately 600 g of Erythromycin resistance and Gram-positive origin of replication. A 5.0 kbp fragment was isolated by electroelution from a 0.5 V agarose gel.

In the same manner, the HindIII fragment bearing the desulphohirudin gene was isolated from your plasmid pSC12HIR.

The two fragments were linked by T4 ligase and directly transformed with L. lactis LM0230 cells by electroporation with the ligation mixture. Erythromycin resistant transformants were selected and prepared from the ρ1 plasmid DNA.

Restriction analysis of the resulting plasmids confirmed the desired structure: both expression orientations were constructed and could be used to produce desulphohirudin in Bacillus thuringiensis. The plasmids were designated pVAHIR and pVAHIR-1.

• · · · • · · · · • · · · • ·»·· · • · · · ·«·· ··

5.2. Production of desulphohirudin in B. thuringiensis

B. thuringiensis strain HDlcryB [DSM 4547; Schurter et al. (1989) were transformed with pVAHIR plasmid by electroporation.

Transformants were selected on LB plates containing 20 µg / ml erythromycin at 27 ° C or 30 ° C. The? 1 plasmid DNA was extracted from the transformants as described for L. lactis; restriction analysis confirmed that the plasmid isolated was identical to your pVAHIR plasmid.

Transformants carrying pVAHIR or pV838 as a control were cultured in LB medium containing 20 mg / L erythromycin at 27 ° C overnight, and the culture was diluted 1: 200 with fresh medium and further incubated at 70 ° C. Seven hours after dilution, the culture was centrifuged and the supernatant desulfohirudin activity was measured.

Desuzolphiririn was detected in the culture supernatant of cells transformed with plasmid pVAHIR, whereas no activity was detected in the control transformed with plasmid pVA838.

Deposited microorganisms

In accordance with the Budapest Convention, the following strains have been deposited with the Deutsche Sammlung von Mikroorganismen • ·

<img file="HUT57263A_D0023.tif" />

- 61 und Zellkulturen (DSM), in the collection of Mascheroder Weg lb, D-3300 Braunschweig:

<td>Escheri chia</td><td colspan="2">coli K12 TGl / pUCRS</td><td>DSM</td><td> 5803</td><td> 1990 .</td><td> 02 .</td><td> 16</td>

<td>Lactococcus</td><td>lactis</td><td>LL712</td><td>DSM</td><td> 5804</td><td> 1990 .</td><td> 02.</td><td> 16</td>

<td>Lactococcus</td><td>lactis</td><td>LM0230</td><td>DSM</td><td> 5805</td><td> 1990.</td><td> 02 .</td><td> 16</td>

Literature

Bates EM et al., Appl. Environm. Microbiol. 55, 2095 (1989).

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

My buds HC and Doly J., Nucl. Acids Slit. 7, 1513 (1979).

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

Clewell D. and Helsinki DR, Proc. Natl. Acad. Sci. USA 62, 1159 (1969).

Davies FL, Underwood HM and Gasson JM, J. Appl. Bacteriol. 51, 325 (1981).

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

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

Gasson JM, J. Bacteriol. 154, 1 (1983).

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

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

Jós Μ. , Appl. Environ. Microbiol. 50, 540 (1985).

Karn, J., et al., Proc. Natl. Acad. Sci. USA 77, 5172 (1980), · · · · · · · · · · · · · · · · · · ··· ··

Laemmli UK. Natura 227. 680 (1970)

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

Maniatis T., Fritsch EF, and Sarnbrook J. Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory.

New York (1982)

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

Norris K. et al. Nucl. Acids Slit. 11, 5103 (1983).

Powell et al., Appl. Environm. Microbiol. 54, 6 (1988)

Oto R. et al. Appl. Environm. Microbiol. 43, 1272 (1982).

Rusche JR and Howards-Flanders P., Nucl. Acids Slit. 13, 1997 (1985).

S'anger F., Miiken S. and Coulson AR. Proc. Natl. Acad. Sci. USA 74, 5463 (1977).

Schurter W., Geiser M. and Mathe D. Gene. Genet. 218, 177 (1989).

Terzaghi BK and Sadine NE, Appl. Microbiol. 29, 807 (1975) Von Heijne G., Eur. J. Biochem. 133, 17 (1983).

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

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

<img file="HUT57263A_D0024.tif" />

- 63 Gene sequences

Type: nucleotide sequence with the sequence of the corresponding protein

Length: 1920 base pairs

Thread type: double

Topology: linear

Type of molecule: genomic DNA

Original organization: Lactococcus lactis LM0230 (DSM 5805)

Direct experimental source: plasmid pUCRS (DSM 5803)

The site of the genome is also chromosomes

Characteristics: 1-400 bp: part of the promoter region

411 - 491 bp: MSP signal peptide

492 - 1793 bp is the mature MSP protein

Function: gene of L. lactis major secretion product (MSP)

<td>TTTAGGTATT</td><td>TACGGAATTG</td><td>CGACCTTATT</td><td>GTTCCCACTT</td><td> 40</td>

<td>ATTGCTCTTT</td><td>TTGTATATAA</td><td>TATACAAATA</td><td>ACTATATTTA</td><td> 80</td>

<td>CTAATCGCTG</td><td>GACAAGGCTT</td><td>TTTACAACAA</td><td>TTATTATTGT</td><td> 120</td>

<td>GACCGCTTTT</td><td>GAAGTTTTTA</td><td>GTGCAATCAT</td><td>TATGACAGCT</td><td> 160</td>

<td>TTTGGATTTG</td><td>CCCAACTTCA</td><td>GTTTATCAAA</td><td>TTTGTTGTTT</td><td> 200</td>

<td>ACCAGTTAGC</td><td>GCCTACACTT</td><td>TTGCTCAATA</td><td>TTATCTTAGC</td><td> 240</td>

<td>TGTAGCCTTA</td><td>CAATTCCCTT</td><td>TAGAAATCTT</td><td>TTACAGATTA</td><td> 280</td>

<td>AAGAAAAGTC</td><td>ATGTAAGATA</td><td>CAATTAGAAA</td><td>GTGTTTTGTA</td><td> 320</td>

i

<img file="HUT57263A_D0025.tif" />

- 64 ATCATAAAGA AATATTAAGG TGGGGTAGGA ATAGTATAAT

ATGTTTATTC AACCGAACTT AATGGGAGGA AAAATTAAAA

360

400

AAGAACAGTT ATG AAA AAA AAG ATT ATC TCA GCT 434

Met Lys Lys Lys Ile Ile Ser Alá

<td>ATT</td><td>TTA</td><td>ATG</td><td>TCT</td><td>ACA</td><td>GTG</td><td>OVER THE</td><td>CTT</td><td>TCT</td><td>GCT</td><td>GCA</td><td> 467</td>

<td>Ile</td><td>Leu</td><td>Met</td><td>Ser</td><td>Thr</td><td>With</td><td>Ile</td><td>Leu</td><td>Ser</td><td>below</td><td>below</td><td></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></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>ACA</td><td>AAC</td><td> 500</td>

<td>below</td><td>Pro</td><td>Leu</td><td>Ser</td><td>Gly</td><td>With</td><td>Tyr</td><td>below</td><td>asp</td><td>Thr</td><td>Asn</td><td></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></td>

<td>TCA</td><td>DAM</td><td>ATT</td><td>GCT</td><td>AAA</td><td>CAA</td><td>DAM</td><td>GCG</td><td>ACA</td><td>ATT</td><td>TCA</td><td> 533</td>

<td>Ser</td><td>asp</td><td>Ile</td><td>below</td><td>Lys</td><td>Gin</td><td>asp</td><td>below</td><td>Thr</td><td>Ile</td><td>Ser</td><td></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></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> 566</td>

<td>Ser</td><td>below</td><td>Gin</td><td>Ser</td><td>below</td><td>Lys</td><td>below</td><td>Gin</td><td>below</td><td>Gin</td><td>below</td><td></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></td>

<td>CAA</td><td>GTT</td><td>DAM</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> 599</td>

<td>Gin</td><td>With</td><td>asp</td><td>Ser</td><td>Leu</td><td>Gin</td><td>Ser</td><td>Lys</td><td>With</td><td>asp</td><td>Ser</td><td></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></td>

<td>TTA</td><td>CAA</td><td>CAA</td><td>AAG</td><td>CAA</td><td>ACA</td><td>AGT</td><td>ACT</td><td>AAA</td><td>GCA</td><td>CAA</td><td> 632</td>

<td>Leu</td><td>Gin</td><td>Gin</td><td>Lys</td><td>Gin</td><td>Thr</td><td>Ser</td><td>Thr</td><td>Lys</td><td>below</td><td>Gin</td><td></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></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> 665</td>

<td>Ile</td><td>below</td><td>Lys</td><td>Ile</td><td>Glu</td><td>Ser</td><td>Glu</td><td>Arg</td><td>Lys</td><td>below</td><td>Leu</td><td></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></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> 698</td>

<td>Asn</td><td>below</td><td>Gin</td><td>Ile</td><td>below</td><td>Thr</td><td>Leu</td><td>Asn</td><td>Glu</td><td>Ser</td><td>Ile</td><td></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></td>

<td>AAA</td><td>GAA</td><td>CGT</td><td>ACA</td><td>AAG</td><td>ACA</td><td>TTG</td><td>GAA</td><td>GCT</td><td>CAA</td><td>GCA</td><td> 731</td>

<td>Lys</td><td>Glu</td><td>Arg</td><td>Thr</td><td>Lys</td><td>Thr</td><td>Leu</td><td>Glu</td><td>below</td><td>Gin</td><td>below</td><td></td>

<td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td>

• ·· ·

- 65 • · ·· ·· • · · · * 9· • · · · ♦··♦ « ♦··· · · &lt; · · ·«· · ··«· 9 9· ·

<td>CGT</td><td>AGT</td><td>GCT</td><td>CZXA</td><td>GTT</td><td>ZXAC</td><td>AGC</td><td>TCA</td><td>GCA</td><td>ACA</td><td>ZXAT</td><td> 764</td>

<td>Arg</td><td>Ser</td><td>below</td><td>Gin</td><td>With</td><td>Asn</td><td>Ser</td><td>Ser</td><td>below</td><td>Thr</td><td>Asn</td><td></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></td>

<td>TAT</td><td>ATG</td><td>DAM</td><td>GCT</td><td>GTT</td><td>GTT</td><td>ZXAT</td><td>TCA</td><td>ZXZXA</td><td>TCT</td><td>TTG</td><td> 797</td>

<td>Tyr</td><td>Met</td><td>asp</td><td>below</td><td>With</td><td>With</td><td>Asn</td><td>Ser</td><td>Lys</td><td>Ser</td><td>Leu</td><td></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></td>

<td>ACA</td><td>DAM</td><td>GTT</td><td>ATT</td><td>CZXA</td><td>ZXZXA</td><td>GTA</td><td>ACA</td><td>GCT</td><td>ATT</td><td>GCT</td><td> 830</td>

<td>Thr</td><td>asp</td><td>With</td><td>Ile</td><td>Gin</td><td>Lys</td><td>With</td><td>Thr</td><td>below</td><td>Ile</td><td>below</td><td></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></td>

<td>ACT</td><td>GTT</td><td>TCT</td><td>AGT</td><td>GCC</td><td>ZXAC</td><td>ZXZXA</td><td>CZXA</td><td>ATG</td><td>TTG</td><td>GZXA</td><td> 863</td>

<td>Thr</td><td>With</td><td>Ser</td><td>Ser</td><td>below</td><td>Asn</td><td>Lys</td><td>Gin</td><td>Met</td><td>Leu</td><td>Glu</td><td></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></td>

<td>CAA</td><td>CAA</td><td>GZXA</td><td>ZXZXA</td><td>GAG</td><td>CZXA</td><td>ZXZXA</td><td>GAG</td><td>CTT</td><td>AGC</td><td>CZXA</td><td> 896</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>Leu</td><td>Ser</td><td>Gin</td><td></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></td>

<td>AAG</td><td>TCA</td><td>GZVA</td><td>ACT</td><td>GTT</td><td>ZXAA</td><td>ZXAG</td><td>ZXAC</td><td>TAC</td><td>ZXAC</td><td>CAG</td><td> 929</td>

<td>Lys</td><td>Ser</td><td>Glu</td><td>Thr</td><td>With</td><td>Lys</td><td>Lys</td><td>Asn</td><td>Tyr</td><td>Asn</td><td>Gin</td><td></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></td>

<td>TTC</td><td>GTT</td><td>TCT</td><td>CTT</td><td>TCA</td><td>CZXA</td><td>AGT</td><td>TTG</td><td>DAM</td><td>TCT</td><td>CZXA</td><td> 962</td>

<td>Phe</td><td>With</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Gin</td><td>Ser</td><td>Leu</td><td>asp</td><td>Ser</td><td>Gin</td><td></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></td>

<td>GCT</td><td>CAA</td><td>GZXA</td><td>TTG</td><td>ACT</td><td>TCA</td><td>CZXA</td><td>CZXA</td><td>GCT</td><td>GZXA</td><td>CTC</td><td> 995</td>

<td>below</td><td>Gin</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Ser</td><td>Gin</td><td>Gin</td><td>below</td><td>Glu</td><td>Leu</td><td></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></td>

<td>AAA</td><td>GTT</td><td>GCG</td><td>ACT</td><td>TTG</td><td>ZXAC</td><td>TAT</td><td>CZXA</td><td>GCA</td><td>ACA</td><td>ATT</td><td> 1028</td>

<td>Lys</td><td>With</td><td>below</td><td>Thr</td><td>Leu</td><td>Asn</td><td>Tyr</td><td>Gin</td><td>below</td><td>Thr</td><td>Ile</td><td></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></td>

<td>GCA</td><td>ACT</td><td>GCG</td><td>CZXA</td><td>DAM</td><td>ZXZXA</td><td>ZXZXA</td><td>CZXA</td><td>GCT</td><td>TTA</td><td>TTA</td><td> 1061</td>

<td>below</td><td>Thr</td><td>below</td><td>Gin</td><td>asp</td><td>Lys</td><td>Lys</td><td>Gin</td><td>below</td><td>Leu</td><td>Leu</td><td></td>

<td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td>

·· ····

<td>DAM</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> 1094</td>

<td>asp</td><td>Glu</td><td>Lys</td><td>below</td><td>below</td><td>below</td><td>G1U</td><td>Lys</td><td>below</td><td>below</td><td>Gin</td><td></td>

<td></td><td></td><td> 22 5</td><td></td><td></td><td></td><td></td><td> 230</td><td></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> 1127</td>

<td>Glu</td><td>below</td><td>below</td><td>Lys</td><td>Lys</td><td>Gin</td><td>below</td><td>below</td><td>Tyr</td><td>Glu</td><td>below</td><td></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></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> 1160</td>

<td>Gin</td><td>Gin</td><td>Lys</td><td>Glu</td><td>below</td><td>below</td><td>Gin</td><td>below</td><td>Gin</td><td>below</td><td>below</td><td></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></td>

<td>TCA</td><td>ACA</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> 1193</td>

<td>Ser</td><td>Thr</td><td>below</td><td>below</td><td>Thr</td><td>below</td><td>Lys</td><td>below</td><td>With</td><td>Glu</td><td>below</td><td></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></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> 1226</td>

<td>below</td><td>Thr</td><td>Ser</td><td>Ser</td><td>below</td><td>Ser</td><td>below</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Gin</td><td></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></td>

<td>GCT</td><td>CCA</td><td>CAA</td><td>GTA</td><td>AGT</td><td>ACA</td><td>AGC</td><td>ACT</td><td>DAM</td><td>AAT</td><td>ACA</td><td> 1259</td>

<td>below</td><td>Pro</td><td>Gin</td><td>With</td><td>Ser</td><td>Thr</td><td>Ser</td><td>Thr</td><td>asp</td><td>Asn</td><td>Thr</td><td></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></td>

<td>ACA</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> 1292</td>

<td>Thr</td><td>Ser</td><td>Asn</td><td>below</td><td>Ser</td><td>below</td><td>Ser</td><td>Asn</td><td>Ser</td><td>Ser</td><td>Asn</td><td></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></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> 1325</td>

<td>Ser</td><td>Ser</td><td>Ser</td><td>Asn</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Ser</td><td></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></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> 1358</td>

<td>Ser</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Asn</td><td>Ser</td><td></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></td>

<td>AAT</td><td>GCT</td><td>GGT</td><td>GGG</td><td>AAT</td><td>ACA</td><td>AAT</td><td>TCA</td><td>GGC</td><td>ACT</td><td>AGT</td><td> 1391</td>

<td>Asn</td><td>below</td><td>Gly</td><td>Gly</td><td>Asn</td><td>Thr</td><td>Asn</td><td>Ser</td><td>Gly</td><td>Thr</td><td>Ser</td><td></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></td>

<td>ACT</td><td>GGA</td><td>AAT</td><td>ACT</td><td>GGA</td><td>GGA</td><td>ACA</td><td>ACT</td><td>ACT</td><td>GGT</td><td>GGT</td><td> 1424</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><td></td>

<td></td><td></td><td> 335</td><td></td><td></td><td></td><td></td><td> 340</td><td></td><td> »</td><td></td><td></td>

♦ we., · · 4 ··

<td>AGC</td><td>GGT</td><td>OVER THE</td><td>AAT</td><td>AGT</td><td>TCA</td><td>CCA</td><td>ATT</td><td>GGA</td><td>AAT</td><td>CCT</td><td> 1457</td>

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

<td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td><td></td><td></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>TAT</td><td>GTA</td><td>TGG</td><td> 14 90</td>

<td>Tyr</td><td>below</td><td>With</td><td>Gly</td><td>Gly</td><td>Cys</td><td>Thr</td><td>asp</td><td>Tyr</td><td>With</td><td>Trp</td><td></td>

<td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</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>TAT</td><td>ATC</td><td>AGA</td><td> 1523</td>

<td>Gin</td><td>Tyr</td><td>Phe</td><td>below</td><td>below</td><td>Gin</td><td>Gly</td><td>Ile</td><td>Tyr</td><td>Ile</td><td>Arg</td><td></td>

<td></td><td></td><td></td><td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</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>CAA</td><td>TGG</td><td>GCT</td><td> 1556</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>Gin</td><td>Trp</td><td>below</td><td></td>

<td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td><td> 385</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>CTC</td><td>CAT</td><td>GTT</td><td> 1589</td>

<td>Ser</td><td>Asn</td><td>Gly</td><td>Pro</td><td>below</td><td>Gin</td><td>Gly</td><td>With</td><td>Leu</td><td>His</td><td>With</td><td></td>

<td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></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>GCA</td><td>TCA</td><td>AGC</td><td> 1622</td>

<td>With</td><td>Gly</td><td>below</td><td>below</td><td>Pro</td><td>Gly</td><td>With</td><td>Ile</td><td>below</td><td>Ser</td><td>Ser</td><td></td>

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

<td>TTC</td><td>TCA</td><td>GCT</td><td>DAM</td><td>TTT</td><td>GTT</td><td>GGA</td><td>TAT</td><td>GCA</td><td>AAC</td><td>TCA</td><td> 1655</td>

<td>Phe</td><td>Ser</td><td>below</td><td>asp</td><td>Phe</td><td>With</td><td>Gly</td><td>Tyr</td><td>below</td><td>Asn</td><td>Ser</td><td></td>

<td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td><td></td><td></td><td></td><td> 420</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>AJKA</td><td>TCA</td><td>GTT</td><td> 1688</td>

<td>Pro</td><td>Tyr</td><td>Gly</td><td>His</td><td>With</td><td>below</td><td>Ile</td><td>With</td><td>Lys</td><td>Ser</td><td>With</td><td></td>

<td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>AAT</td><td>TCA</td><td>DAM</td><td>GGT</td><td>ACA</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>Ser</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>ACA</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>With</td><td>Ser</td><td>below</td><td>Ser</td><td>Gly</td><td>With</td><td>Thr</td><td>Phe</td><td>Leu</td><td>Met</td><td></td>

455 460

<img file="HUT57263A_D0026.tif" />

• 4 ····

<img file="HUT57263A_D0027.tif" />

1826

CCA AAC TAG AAAAAAGTCT TAATAAATAA AAAATAGTGG

Pro Asn

465

TTTGATAGTG GGGAATAATT TTCCTTCTGT CAAATCATTT

1866

TTTATTATTG TGGTATAATA ATAAGGAAAA ATGATAAGGG

1906

GATAGATACA AATG

1920

SEQ ID No. 2

Type: nucleotide sequence with the corresponding polypeptide sequence

Length: 279 bp

Thread type: double. .

Topology: linear

Molecule Type: Recombinant

Original Source: Lactococcus lactis LM0230 (DSM 5805);

Hirudo's medicine, too

Direct experimental source: plasmid pUCRS (DSM 5803);

Plasmid PML310 (EP-A 168 342)

Characteristics: 1 to 81 bp: a marker peptide of MSP

- 279 bp: coding region for desulfo-hirudin

Function: A fusion gene composed of DNA encoding the MSP signaling peptide of L. lactis Lm0230 and the structural gene of hirudin for the production of secreted hirudin in bacteria.

-&lt; · (

-· (

<td>ATG Met</td><td>AAA Lys</td><td>AAA Lys -25</td><td>AAG Lys</td><td>ATT Ile</td><td>ATC Ile</td><td>TCA Ser</td><td>GCT below -20</td><td>ATT Ile</td><td>TTA Leu</td><td>ATG Met</td><td>TCT Ser</td><td> 36</td>

<td>ACA</td><td>GTG</td><td>OVER THE</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>With</td><td>Ile</td><td>Leu</td><td>Ser</td><td>below</td><td>below</td><td>below</td><td>Pro</td><td>Leu</td><td>Ser</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>With</td><td>Tyr</td><td>below</td><td>With</td><td>With</td><td>Tyr</td><td>Thr</td><td>asp</td><td>Cys</td><td>Thr</td><td>Glu</td><td>Ser</td><td></td>

<td></td><td></td><td></td><td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td></td>

<td>GGT</td><td>CAG</td><td>AAC</td><td>CTG</td><td>TGC</td><td>CTG</td><td>TOC</td><td>GAA</td><td>GGT</td><td>TCT</td><td>AAC</td><td>GTT</td><td> 144</td>

<td>Gly</td><td>Gin</td><td>Asn</td><td>Leu</td><td>Cys</td><td>Leu</td><td>Cys</td><td>Glu</td><td>Gly</td><td>Ser</td><td>Asn</td><td>With</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> 20</td><td></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>Cys</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Asn</td><td>Lys</td><td>Cys</td><td>Ile</td><td>Leu</td><td>Gly</td><td>Ser</td><td>asp</td><td></td>

<td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></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>Gly</td><td>Glu</td><td>Lys</td><td>Asn</td><td>Gin</td><td>Cys</td><td>With</td><td>Thr</td><td>Gly</td><td>Glu</td><td>Gly</td><td>Thr</td><td></td>

<td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td>

<td>CCG</td><td>AAA</td><td>CCG</td><td>CAG</td><td>TCT</td><td>CAC</td><td>AAC</td><td>GAC</td><td>GGT</td><td>GAC</td><td>TTC</td><td>GAA</td><td> 252</td>

<td>Pro</td><td>Lys</td><td>Pro</td><td>Gin</td><td>Ser</td><td>His</td><td>Asn</td><td>asp</td><td>Gly</td><td>asp</td><td>Phe</td><td>Glu</td><td></td>

<td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></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>BROAD</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>Leu</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="HUT57263A_D0028.tif" />