Improved recombinant hosts, their production and use.

The invention relates to the recombinant production of heterologous proteins under regulatable promoter control. More specifically, the invention relates to providing hosts with at least one modification in the genome associated with aporepressor-activator production. The presence of the modification permits more effective production of the desired protein.

Feedback control systems which regulate the metabolic pathways of both procaryotic and eucaryotic organisms have been known for many years to exist both at the genetic and protein levels. Perhaps the best known examples of genetic level feedback control are regulatable promoter/operator systems, such as the β-galactosidase (lac) promoter/operator and the tryptophan (trp) promoter/operator systems in procaryotic hosts. The lac operator responds to a constitutive protein repressor to prevent transcription. In the presence of an inducer such as lactose or IPTG, the repressor is bound to inducer, and unable to bind to the operator, thus transcription is permitted. The trp operator, conversely, responds only to an "activated" repressor protein; i.e., in the absence of tryptophan, the repressor does not bind to operator. Advantage has been taken of these genetic level controls to effect the production of recombinant proteins at times of the host cells' life cycle which are advantageous to efficient production.

Typically, the coding sequence for a desired heterologous protein is ligated in operable configuration with the regulatable promoter/operator in suitable vectors to transform the host. Since immediate production of the heterologous protein during the growth phase of the transformed organism would be injurious to the healthy multiplication of the bacteria, this production is delayed by causing production of sufficient repressor to keep the promoter/operator system turned off.

In the case of the trp operator, the mediator of the on/off switch is the combination of tryptophan with a repressor protein (aporepressor) produced normally by the host, but at a level dependent on the concentration of tryptophan in the medium. Combination with tryptophan permits the aporepressor to function, and expression of coding sequences under control of the trp promoter is delayed by the presence of tryptophan until further repression is no longer needed. Under usual recombinant host growth conditions, derepression is effected either by depleting the supply of tryptophan in the medium or by supplying an antagonist to tryptophan such as indole acrylic acid (IAA), which interferes with the ability of tryptophan to bond to the aporepressor.

While, therefore, in principle, the trp promoter system offers a convenient means to delay expression of heterologous gene sequences until a desired point in the growth curve is reached and then permit operation of the promoter, this control is not absolute. One difficulty is that complete depletion of the tryptophan is impractical because tryptophan is a component of many proteins, and is required at some level to enable protein synthesis. The result is that the trp promoter is always partially repressed due to this minimal tryptophan level.

The mechanisms for regulating tryptophan production have been studied by C. Yanofsky and his colleagues for many years. Studies conducted more than twenty years ago provided a revertant of a tryptophan-requiring mutant to partial tryptophan independence. This partial revertant apparently exhibited higher expression levels of the gene sequences under trp promoter control, but exhibited a slower growth rate without tryptophan than does wild type. A study of this s mutant, A46PR9, (Henning, U., et al., Proc Natl Acad Sci (USA) (1962) 48:1497-1504), showed that a mutation in the coding sequence for trpA, an enzyme which catalyzes one of the steps in the conversion of indole glycerol phosphate to tryptophan, contains a mutation which results in the substitution of a valine residue for a glycine residue. The result is a protein which is less active in performing this catalysis than the native protein, thus accounting for the slow rate of growth in the absence of tryptophan. On the other hand, higher level amounts of this relatively inactive protein are formed, thus indicating a higher degree of expression for the genomic sequences under control of the trp promoter/operator.

The importance of this trp operon regulation for enhanced expression is further elucidated in a more recent paper of the same group (Kelley, R. L., et al., Proc Natl Acad Sci (USA) (1982), 79:3120-3124). LacZ fusion flags were used to show that the response of a typical procaryotic cell to variations in tryptophan concentrations is regulated mainly by the trp operon, which shows a 70-fold variation over the tryptophan concentration range studied, whereas the trp aporepressor synthesis showed a variation of only 4-5 fold.

Since, therefore, the level of production of heterologous proteins under trp control in recombinant hosts depends on the ability of the trp promoter to exhibit a high level of efficiency in promoting transcription, it. would be desirable to introduce, in recombinant hosts, alterations which would result in more dramatic derepression. From results with A46PR9 it would appear that the coding sequence for the modified trpA protein results in enhanced expression levels effected by the trp promoter operably linked to its own coding sequence. The effect could be applicable with regard to any trp promoter in the cell, including a trp promoter in operable linkage with the recombinant heterologous coding sequences. Therefore, hosts having this alteration in the genome may be high producers when transformed with vectors using trp control.

Analogous host cell modifications affecting promoters susceptible to similar feedback control mechanisms are also desirable in instances where these promoters can be used to control expression of heterologous proteins. For example, the phenylalanine A (pheA) promoter/operator and tyrosine (aroF) promoter/operator are less frequently used for recombinant expression. However, their sequences and mode of regulation are known, and their use for the production of heterologous proteins is within the skill of the art. (See Hudson, G. S., et al., J Mol Biol (1984) 180:1023-1051.)

Such host cell improvements are the subject of the present invention.

The present invention provides recombinant procaryotic hosts with the ability to permit more efficient expression of heterologous- coding sequences under the control of regulatable promoters. These hosts may be prepared by altering the genome of the host organism to obtain mutant sequences under control of the same promoter as that used for the expression of the recombinant DNA sequence, which mutant sequences result in increased levels of their own expression. Typically the mutant sequences encode enzymes responsible for the synthesis of an aporepressor-activator. The result is that derepression of expression is enhanced.

Thus, in one aspect, the invention is directed to a recombinant host -cell with diminished ability to synthesize an a^porepressor-activator. This host cell has an alteration in its genome resulting in enhanced expression under the control of a promoter which can also be used to control recombinant protein production in the same host. Another aspect of the invention is a protein expression system comprising the foregoing host transformed with a vector containing the coding sequence for a desired protein under the control of a promoter/operator which is responsive to the same aporepressor-aporepressor-activator as is the operon which is modified so as to be more effectively derepressed.

Brief Description of the Drawings

<ul id="ul0001" list-style="none"><li>Figure 1 shows a diagram of the process for KB2 production using Pl phage containing cysB^-, Tn5 and Pl phage containing. trpA46PR9.</li><li>Figure 2 shows the construction of the expression vector for IL-2, pLW45.</li><li>,. Figure 3 shows stained SDS-gels run on extracts from pLW45 transformed hosts.</li></ul>

Modes of Carrying Out the Invention

A. Definitions

As used herein, "aporepressor-activator" refers to a substance which combines with an aporepressor protein. The combination is effective in Inhibiting expression under the control of a target promoter.

In a commonly encountered form of transcription control, the promoter is provided with an operator sequence, which, when bound with a suitable repressor, shuts off the transcription under the subject promoter's control. The repressor may be a protein which is produced under the control of an entirely different promoter elsewhere in the cell's DNA which is, by itself, capable of combining with the operator sequence to shut off the subject gene. This is the situation with respect to the lac (p-galactosidase) promoter/operator, which is controlled by a repressor protein produced under the control of a different promoter. However, a number of other control systems require that the aporepressor protein be activated by combination with another substance--the "aporepressor-activator". Typically this substance is the end product of the synthetic sequence whose production is controlled by the subject gene. Familiar examples are the trp promoter, where the aporepressor (trpR) must be combined with tryptophan in order to repress; tyrosine (aroF) promoter, which controls the synthesis of enzymes in the pathway to tyrosine, which is regulated by an aporepressor (tyrR) in combination with tyrosine; and the analogous operon controlling the production of enzymes required in the synthesis of phenylalanine where the aporepressor also requires combination with the end product to be effective. Thus, "aporepressor-activator" refers to those substances which must be available to combine with the aporepressor protein in order to control the subject regulatable promoter. <ul id="ul0002" list-style="none"><li>"Repressor" refers to a substance effective in this regard; it may be an aporepressor (protein) alone or said protein in combination with the aporepressor-activator.</li><li>"Regulatable promoter" refers to a promoter whose operation is controlled by the presence or absence of a substance in a cell or cell culture such as a repressor, or by other environmental conditions such as temperature, salt concentration, and so forth.</li><li>"Operably linked" refers to juxtaposition wherein the functionality of the operably linked subjects are preserved. Thus, promoter operably linked to a coding sequence may result in expression of the coding sequence under control of the promoter under proper conditions.</li><li>"Cells", "cell cultures", "host cells", "recombinant host cells" refer to subject cells for recombinant DNA manipulations. As would be apparent from the context, these cells may be candidates for, or resultants of, transfer of new DNA sequences according to recombinant techniques. Techniques which are suitable for DNA uptake by cells include, most prominently, in vitro transformation; however, other techniques such as transduction or conjugation may also be used. The definition further includes the progeny of the cells directly referred to. It is understood that such progeny may not be precisely identical in DNA content to their parents, but such progeny are included in the definition so long as alterations due, for example, to accidental or deliberate mutation do not destroy the ability of the cells to exhibit the properties conferred by the DNA introduced in a manner similar to that exhibited by their parents.</li></ul>

B. General Description

Any mutant host, produced by whatever means, having a genomic modification which results in increased expression (as compared to wild type) of sequences under control of a regulatable promoter when the promoter is derepressed is a candidate host for the expression systems of the invention. For example, in the illustration below, E. coli trp A46PR9 could, itself, be used as a host. If the organism has, however, other characteristics which make it less desirable as a recombinant host, it may nevertheless be used as a donor system for the genomic modification as is the case for E. eoti trp A46PR9 in the illustration below. For example, a standard recombinant production strain of E_. coli can be modified to contain mutations in its genome which increase the level of expression for an aporepressor activator operon by first selecting these suitable mutants of a donor strain, and then using a standard phage transduction technique to introduce the desired mutation. In the alternative, the production strain may be subjected to mutation pressure through irradiation or chemical treatment, and desirable mutants selected. The host, whose genomic sequences have been altered in the aporepressor activator operon, may then be used to produce recombinant proteins by introducing a vector which contains an expression system for the desired recombinant protein under the control of the same regulatable promoter as that controlling the aporepressor-activator operon.

While E. coli hosts are exemplified below, and are clearly the most convenient for routine production, other forms of procaryotic hosts are clearly not excluded from the invention. Therefore, strains of bacillus, for example, Bacillus subtilis or of Pseudomonas, might also be used as subjects for constructing the host organisms useful in the invention. Similarly, while the trp promoter/operator and trp operon are exemplified, any operon system which is controlled analogously could be used. Thus, for example, the operons controlling production of tyrosine or phenylalanine may also be used.

In general, in a convenient method to prepare the host, the desired substrate host is first infected with general transducing phage stock obtained from lysis of an infected corresponding bacterium containing a marker mutation which maps on the genome proximal to the locus of the desired change. The successfully transduced hosts, now containing the marker, are than transduced with a general transducing phage stock which has been obtained by lysis of cells which contain the desired mutation. The disappearance of the marker can then be used to select for transductants with a high probability of integration of genomic sequences containing the desired mutation. The resulting desired transductants are then suitable as hosts for the recombinant sequences containing the corresponding regulatable promoters.

C. Examples

The following example is intended to illustrate, but not to limit, the invention. The example uses an alteration of the trp operon which is transduced into a convenient E. coli recombinant host strain by the aforementioned phage transduction techniques. The construction of the host KB2 is shown in Figure 1.

: E. coli strain W3110 cysB^-, Tn5 is used as a donor strain for the marker sequences. The cysB^- locus is at approximately 28.0' on the E. coli genome, which is proximally adjacent to the trp operon locus at 27.7'. The Tn5 transposon in the donor strain is proximal to the cysB^- also, and the presence of Tn5 results in resistance to the antibiotics kanamycin, neomycin, and other aminoglycosides. The presence of the closely linked Tn5 permits selection of transductants using resistance to these antibiotics.

Accordingly, E. coli W3110 (cysB-, Tn5) is infected with the standard transducing phage PI and the resulting lysates used to transduce the recombinant production strain E. coli K12 MM294-1. The transduction is performed essentially as described by Lennox, E. S., Virology (1955) 1:190, as modified by Yanofsky, et a1., Virology (1959) 8:425, and Maling, R. D., et al., Proc Natl Acad Sci (Wash) (1964) 47:551.

The transduced cells were then plated on media containing 30 µg/ml kanamycin to select for Kan^R. Successful colonies were screened for cysB^-, i.e., a growth requirement for cysteine, and approximately 20% of the kanamycin-resistant (Kan^R) colonies showed this characteristic. One such colony, designated KB1, was selected as the substrate host.

E. coli KB1 was then transduced using lysates of Pl-infected E. coli trpA46PR9, obtained from Professor Yanofsky at Stanford University, a strain generally available upon request. This donor strain is wild type with respect to cysB. The transductant cultures obtained from KB1 using these lysates were then selected for cysB⁺ by... growth in medium lacking cysteine. The selected cultures were screened for kanamycin sensitivity, and approximately 50% of the transductants were kanamycin sensitive. Finally, the kanamycin-sensitive colonies were screened for the presence of the desired mutation by assessing the level of anthranilate synthase (trpE gene product) another enzyme besides tryptophan synthetase (trpA gene product) produced by the trp operon--in colonies grown on glucose- minimal media. Eleven percent of the kanamycin-sensitive colonies showed high levels of this enzyme, and accordingly were apparently more efficient than wild type in producing the protein products of the trp operon.

The resulting successful strain, designated E. coli MM294/A46PR9 KB2, was deposited with ATCC on March 29, 1985 under the provisions of the Budapest Treaty and given accession number 53,075.

E. coli KB2 was transformed using the standard transformation technique for procaryotes of Cohen, S.N. et al., Proc Natl Acad Sci (1972) 69:2110 with pLW45. pLW45, shown in Figure 2, mutein having the cysteine residue at position 125 replaced by a serine. The IL2-coding sequences in pLW45 are under the control of the trp promoter, and the vector confers tetracycline resistance. The pLW45 plasmid may be prepared from two analogous plasmids described in U.S. Patent No. 4,518,584, granted May 21, 1984, assigned to the same assignee and incorporated herein by reference. These plasmids, pLW55 and pLW46, have been deposited with the ATCC and have accession nos. 39,516 and 39,452, respectively, and are now available to the public under the provisions of the Budapest Treaty.

pLW45 is identical to pLW55, except that the N-terminal sequence of the (pLW45) IL-2 lacks an alanine codon of the native sequence. pLW45 contains the desired N-terminal sequence in the same operable linkage with the trp promoter as is found in pLW55. Accordingly, pLW45 is constructed by excising an EcoRI/XbaI fragment from pLW46 (the fragment containing the trp promoter and the N-terminal portion of IL-2) and inserting this fragment into EcoRI/XbaI- digested pLW55.

The transformed hosts were grown in a fermenter containing the following medium: <tables id="tabl0001" num="0001"><img file="EP0197797A2_D0001.tif" /></tables>pH adjusted to 6.50 with 2.5 N NaOH autoclaved.

Sterile Additions (post autoclave) <tables id="tabl0002" num="0002"><img file="EP0197797A2_D0002.tif" /></tables>

Polypropyiene glycol antifoam, glucose, 50% solution, and KOH, 5 N, were added on demand.

The pH of the fermenter was maintained at 6.8 with 5 N KOH. Residual glucose was maintained between 5-10 g/1, dissolved oxygen at 40%, and temperature at 37°C. Harvest was made four hours after the OD₆₈₀ reached about 10. Previous results indicate that tryptophan is depleted and induction occurs at approximately OD₆₈₀=10.

(In other fermenter runs, casamino acids were added to 2% at approximately OD₆₈₀=10, at the start of induction. Under these conditions, the MM294-1 controls produced IL-2 mutein at somewhat higher level (8%-9%) than those indicated below. This additional step was not necessary or helpful in fermentation using MM294/A46PR9.)

Samples of the fermenter culture were removed periodically during the fermentation. Samples were prepared for SDS-PAGE by boiling in SDS, and then applied to gels. The developed gets from the resulting crude extracts were stained with Coomassie blue and scanned with a densitometer. Estimation was thereby nade of the quantity of IL-2 mutein produced both in control cultures using MM294-1 as host and pLW45 as transforming plasmid and in the extracts from pLW45 transformed hosts of the invention. While the induced control organisms produced 3%-5% of total cell protein as IL-2 mutein, the induced transformed cells of the invention produced IL-2 mutein at a level of 18%-20% of total cell protein.

These results are shown in Figure 3. Lane 1 shows proteins from MM294-1 transformed with pLW45 extracted prior to induction; lane 2 shows proteins from these cells after induction; lane 3 shows proteins extracted from induced pLW45-transformed E. coli MM294/A46PR9 (KB2).

The recombinant host system of the present invention may be used in the expression of a wide variety of heterologous proteins or polypeptides. In this connection, the recombinant host system may be used to express proteins or polypeptides such as interferons (alpha, beta, or gamma), lymphokines (interleukins 1, 2 or 3, colony stimulating factors G, GM or 1, tumor necrosis factor) lymphotoxins, leukoregulin, hormones (e.g., insulin), vaccines, enzymes, growth factors, and muteins of these proteins and polypeptides.

The deposits above were made pursuant to a contract between the ATCC and the assignee of this patent application, Cetus Corporation. The contract with ATCC provides for permanent availability of the progeny of this cell line to the public on the issuance of the U.S. patent describing and identifying the deposit or the publications or upon the laying open to the public of any U.S. or foreign patent application, whichever comes first, and for availability of the progeny of this cell line to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 0G 638). The assignee of the present application has agreed that if the cell line on deposit should die or be lost or destroyed when cultivated under suitable-conditions, it will be promptly replaced on notification with a viable culture of the same cell line.

While a preferred embodiment of the invention has been described herein, it can be appreciated that various changes and modifications can be made without departing from the spirit of the invention.