EP0148845A1 - Cloning vector, method of constructing such, and method of concentrating and purifying product proteins produced by the cloning vector - Google Patents

Abstract

New cloning vector characterized by repeated inverted DNA sequences between which an active duplication origin is inserted. Use of said cloning vector, when introduced into a host cell, produces a foreign product protein whose signal sequence is derived from a starting point other than the host cell, the signal sequence mediating the membrane transport of the product protein but retaining it bound to the cell surface until released by a change in the environmental conditions in which the host cells find themselves. This system provides a means of regulating the release of product proteins for concentration in a highly purified form.

Description

CLONING VECTOR, METHOD OF CONSTRUCTING SUCH, AND

METHOD OF CONCENTRATING AND PURIFYING PRODUCT PROTEINS

PRODUCED BY THE CLONING VECTOR

Field of Invention

This invention is cross-referenced to com¬ monly assigned U.S. application Serial No. 508,391 filed June 29, 1983 and from which this application claims priority.

This invention relates to stable cloning vec¬ tors involving inverted repeated sequences of DNA be¬ tween which is inserted an active origin of DNA repli¬ cation, to a method of constructing such cloning vec- tors, to the use of transformant cells to produce foreign product protein derived from the stable cloning vectors, and to a method of purifying and concentrating product proteins transported and bound to the cell surface which are produced by-the cloning vectors.

Background Art

Protein Production

The production of foreign proteins by cultured cells containing cloned genes has been described, along with a method of carrying out this procedure, in Cohen and Boyer, U.S. Patent No.

4,237,224. The use of chimeric plas id DNA, as described by Cohen and Boyer, to produce fused protein product capable of being transported to the cell surface has also been described by Silhavy, et al. in

U.S. Patent No. 4,336,336, as have techniques for releasing mature protein products from such a fusion protein cell complex through enzymatic reactions derived from manipulations of the cloned gene, as described by Gilbert et al. in U.S. Patent No.

4,138,397.

OMPI In all examples to date, secretion and subsequent release of foreign fusion protein or mature protein product has been accomplished by utilizing host cell derived signal DNA sequences or hydrophobia leader sequences (HLS) to mediate membrane transport of the product protein. Signal DNA sequences of hydrophobic leader sequences (HLS) are defined as sequences of hydrophobic amino acids at the amino terminus of a polypeptide or protein. Such sequences are located immediately before the structural DNA sequence of a gene for a product polypeptide or protein and after the transitional start signal (ATG) of the gene. These host cell derived signal sequences, as well as signal HLS sequences from closely related strains of bacteria, are similar in that, in all cases, they are responsible for transport of protein molecules through the cell wall or membrane. The signal sequences are clipped off enzymatically by selective cleavage of the signal sequence due to a specific signal peptidase enzyme coded by the bacterial host cell, resulting in release of the product protein from the surface of the cell. Kroyer, Gray and Chang, Genetic Cell Technology 1, 197- 205 (1982); Palva, et al., Proc. Nat. Acad. Sci. 79 (18), 5582-6 (1982). his requirement is fairly restrictive, as signal peptidase enzymes are highly specific in their activity.

No system is known or has been described to date in which signal sequences from more distantly related bacterial cells are used under conditions where membrane transport is mediated by the signal sequences, but appropriate signal peptidase to cleave the product is not present in the host cell. Such a system, described in this application, offers a means of controlling product release for the purpose of concentrating product protein in highly purified form as a part of the harvesting process.

ObΛ?i Cloning Vector

The use of bifunctional chimeric plasmid vectors for expressing foreign proteins is not novel. Constructs similar, but not identical, to pLF119 are described by Horinouchi and Weisblum, J. Bacteriol. 150 (2), 815-25, (1982). Their constructs, as in all other cloned constructs employed for this purpose, avoid the use of inverted repeated segments of DNA in form of palindromic DNA, or transposon structure. In B^ subtilis, E. coli and other bacteria, the presence of inverted repeated DNA sequences has been recognized as a source of instability of the plasmid molecule. Hagan and Warren, Gene 19(1), 147-51 (1982); Hutchison, Sachter and Halvorson, Proc. Intl. Spore Conf., 8th Ed., (1981) Levinson, Am. Soc. Microbiol Press, 123-7. This instability results from the formation of stem- loop structures in the plasmid molecule when complementary strands of adjacent inverted repeated DNA segments form base pairs with each other. Such structures lead to the degradation of the plasmid. This application involves use of such degradation products to generate a novel form of cloning vector.

Disclosure of the Invention The present invention provides a means of constructing and utilizing sequences of DNA which lack homology with chromosomal DNA and which contain inverted repeated (palindromic) sequences of base coding, separated by an active replication origin site, to express foreign genes in bacterial or other cultured cells. Such sequences of DNA may be derived from chimeric plasmids carrying the DNA for the product protein, the signal sequence and plasmid replicative genetic origin material known to be functional in the host cell or in the form of transposable DNA insertion sequences where active terminal repeated DNA sequences

v ^*»^■•:' i ._. flank the DNA for the product protein, the signal sequence, and active replication origin site.

This invention also relates to a method of purifying and concentrating product protein secreted through but bound to the cell wall, with subsequent release of the product protein by change of environmental conditions surrounding the cell. A signal sequence from other than the host cell is used for mediating membrane transport of the product protein but retaining it bound to the cell wall. In particular, the E__j_ coli AMP signal sequence adjacent to the DNA coding for the desired product protein is used to mediate transport of the desired product protein in gram-positive host cells. In these cells, the product protein remains bound to the cell wall until released by change in environmental conditions surrounding the cells. ^' '

Applicants are aware of no work describing the use of gram-negative derived signal sequences in gram-positive organisms. Under these conditions the signal sequence is not cleaved by host cell signal peptidases but is capable of mediating membrane transport of product protein. The product protein is retained bound to the cell wall with subsequent release generated by a change in environmental conditions surrounding the host cells.

In the system discussed membrane transport of the product protein occurs because of similarities between a 23 amino acid signal sequence (see Fig. 5) located at the start of the E^ coli β-lactamase protein and certain polypeptide signal sequences found at the front of proteins which B^ subtilis naturally secretes. The 23 amino acid sequence on the E_^ coli β-lactamase is sufficiently similar to the signal sequences of B^ subtilis to permit it to mediate transport of the protein across the membrane of the B. subtilis cell;

O PI however, the E^ colis signal sequence is not recognized by the enzyme responsible for releasing the protein at the surface of the cell. This results in the protein remaining bound to the cell surface where it accumulates in large quantities as β-lactamase is made and transported by the cell.

Also described is the use of the cloning vector pLF119 (ATCC Deposit Accession No. 39380) and similar vectors which contain inverted repeated DNA sequences to induce over-expression of product protein in gram-positive cells, particularly EL. subtilis and the use of gram-positive bacteria incorporating the cloning vector described to facilitate recovery and release of cell-bound product protein.

Brief Description of the Drawings

Fig. 1 schematically illustrates the method described in the application for preparation of the cloning vector in the form of a chimeric plasmid <pLF119);

Fig. 2 illustrates a map of the chimeric plasmid pLF119 determined by restriction enzyme analysis;

Fig. 3 is a photomicrograph of B^ subtilis strain BR151: Electron microscopy at 4500 X magnification;

Fig. 4 is a photomicrograph of B^ subtilis strain BR151 expressing secreted E__j_ coli β-lactamase product following introduction of pLF119 plasmid DNA: Electron microscopy at 4500 X magnification;

Fig. 5 is the nucleotide sequence of the pBR322 β-lactamase signal or hydrophobic leader sequence;

Fig. 6 shows electrophoresis patterns for proteins recovered following release of E^ coli β- lactamase from the cell surface.

O PI iAr . WIPO Fig. 7 is a graph of amount vs. time for the production of released secreted β-lactamase gene pro¬ duct determined quantitatively by nitrocefin spectro- photometric assay. The E__j_ coli β-lactamase gene pro- duct was produced by B^ subtilis BR151 carrying DNA introduced on plasmid pLF119 grown under various antibiotic pressures compared to control cells receiving similar treatment, including BR151 cells with no external DNA, BR151 cells carrying pC194 plasmid DNA, and BR151 cells carrying pWRlOl plasmid DNA. The spectrophotometric assay was carried out according to the method described in Ross, Methods in Enzy ology (43), 69-85 (1975).

Fig. 8 is a photomicrograph of the "Q" form DNA derived from the plasmid pLF119 denoted as the "quas id" form of DNA derived from plasmid pLF119 in B. subtilis BR151: Electron microscopy at 225,000 X magnification;

Fig. 9 shows an electrophoresis pattern of the DNA band (shown by the arrow) of the "quasmid" illustrated in Fig. 8; and

Fig. 10 describes generation of the "Q" form DNA from inverted DNA sequences flanking a replicative origin.

Best Mode for Carrying Out the Invention

A cloning vector is described which is capable of replicating in host cells, such as E__;_ coli and B_j_ subtilis, and which contains inverted palindromic sequences of base coding separated by an active replication origin site capable of expressing foreign genes in the host cell. The cloning vector may be (1) in the form of chimeric plasmid carrying the DNA for the foreign gene, a signal sequence and plasmid replicative genetic origin material known to be functional in the host cell so that the plasmid is capable of replicating in the host cell and expressing the foreign gene, or (2) in the form of a transposable DNA insertion sequence introduced on plasmid, viral or other DNA vectors. The plasmid vector pBR322 is the most widely used and versatile of the plasmid cloning vectors and contains both ampicillin (AMP) and tetracycline (TΞT) resistance genes and a number of restriction sites. Its complete nucleotide sequence is known. See Maniatis, T., et al.. Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory, 1982. Also see Sutcliff, J. G., "pBR322 Restriction Map Derived from the DNA Sequence: Accurate DNA Size Markers up to 4361 Nucleotide Pairs Long," Nucleic Acid Research 5, 2721, 2728 (1978). The plasmid vector pC194 is also widely used and its complete nucleotide sequence known. Horimuchi and Weisblum, J., J. Bacteriol. 150 (2), 815-25 (1982).

Plasmid pLF119 was constructed, as illustrated in Fig. 1, by ligating a 1.7 Kb Clal fragment of Staph. Aureus plasmid pC194 carrying

Inducible chloramphenicol acetyl transferase and the plasmid replicative origin with inverted repeated copies of 4.3 Kb Clal cleaved pBR322. Alkaline phosphatase treatment was omitted in order to obtain the pBR322 dimer unit. The genetic map of pLF119, showing fragments from which it was constructed, is illustrated by Figs. 1 and 2. Plasmid pLF119 in B. subtilis having ATCC Deposit Accession No. 39380 was deposited June 14, 1983 at the American Type Culture

Collection (ATCC), 12301 Parklawn Drive, Rockville,

Maryland 20852 and then converted to a deposit under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Viability was confirmed. Said deposit is available to the public at the earlier of: grant of a U.S. patent in referenced U.S. application Serial No. 508,391; or continuations or divisions thereof, laying open of this application, publication of this application; or grant of a patent for this invention in any country to the assignee. Selection of the recombinant plasmid was accomplished first in E. coli HB101, selecting for growth on media containing 25 μg/ml of chloramphenicol. Recombinants were then selected for ability to grow on 25 μg/ml a picillin and inability to grow on 25 μg/ml tetracycline.

The structure of the plasmid was confirmed by restriction enzyme mapping. Characteristic Pstl and Hind III cleavage patterns, as well as Hae III, Ace I, and Hinf 1 single and double digests confirmed the structure of the plasmid molecule. The plasmid is stable in E_j_ coli and transforms E^ coli HB101 at a reduced efficiency relative to pBR322. The plasmid is efficiently retained under antibiotic pressure without structural alteration once transformation has been attained and can be amplified to high copy number levels in the E_^ coli host cells.

Plasmid pLF119 DNA isolated from E_j_ coli by cesium chloride density gradient centrifugation effi¬ ciently transforms competent cells or protoplasts of B__;_ subtilis. The transfor ants were screened for acquisi¬ tion of resistance to 25 μg/ml AMP and CAM. Clones which satisfied the antibiotic resistance criteria were grown in PA broth under ampicillin pressure and visually screened for production of pronounced "clumpy" phenotype, as illustrated by Fig. 4.

The use of protoplast fusion techniques to create long linear cells containing many copies of DNA and pooling the DNA from many cells into a multigenic unit increased the amount of E^ coli β-lactamase pro- duced by the cells by several orders of magnitude.

O FI Several techniques may be used to release the product protein from the cell surface in a controlled manner, including mild heating to about 50°-60°C, pH adjust- ment, or alteration of the ionic strength of the culture media.

The DNA sequence of interest for production of the foreign product protein may be cloned into the β-lactamase gene of pBR322 adjacent to the signal sequence end illustrated by Fig. 5. In this application, β-lactamase itself was used as a specific example of a working system; however, other desired proteins or polypeptides may be produced in the same manner by introducing the appropriate DNA sequence into the β-lactamase sequence by linkers or unique cloning sites downstream from the signal sequence region of the gene. To clone in another gene for the β-lactamase gene which is to be expressed in a secretory mode requires that splicing be done so that β-lactamase signal sequence be adjacent the structural gene of interest in the DNA sequence. This can be accomplished by partial digestion of the pLF119 plasmid with the endonuclease Pstl to produce a mixture of molecules cut at the β-lactamase site or sites. Separation of the mixture on agarose gels can be carried out to isolate and extract those DNA molecules representing a single cut sequence. These molecules can then be further treated to introduce cloning sites to allow introduction of foreign genes.

The studies and data, some of which is reproduced hereinafter in the examples, indicate that β-lactamase is produced at constituitive levels in the absence of antibiotic pressure and that ampicillin pressure amplifies expression of the foreign gene product. The foreign gene, in this instance β- lactamase, is secreted by the B^ subtilis cells, remaining bound at the surface of the cells due to the absence of a proper signal peptidase to cleave the pBR322-derived signal sequence. This accumulation of secreted, bound foreign product protein at the cell surface produces the "clumpy" phenotype illustrated in Fig. 4 in comparison to B__j_ subtilis strain BR151 not carrying DNA derived from plasmid pLF119 as illustrated in Fig. 3. Additionally, an anomalous gram stain characteristic was observed for the "clumpy" cells. Heat, pH change, or other change in environmental conditions releases the bound protein product, causing loss of the clumpy phenotype of Fig. 4 and reversion .to normal gram stain traits and appearance, such as illustrated by Fig. 3. At the same time, significant quantities of the free β-lactamase enzyme appear in the supernatant fluid in which the cells are contained.

The "clumpy" cells illustrated in Fig. 4 show the presence of β-lactamase enzyme activity as demon¬ strated by nitrocefin disc assay. The culture supernatant broth did not exhibit β-lactamase activity until exposure of the cells illustrated in Fig. 4 in broth to 50°C for 10 minutes or to pH change. β- lactamase activity then appeared in the supernatant fluid in quantities approaching 5 mg/ml of active pro¬ tein enzyme, as illustrated by Fig. 7. Increased levels of β-lactamase were observed in the presence of ampicillin; however, significant quantities of the enzyme were present in the absence of antibiotic pressure.

SDS gel analysis of the released protein product showed it to be slightly over 30,000 daltons, which is the expected size of the pBR322 coded β- lactamase enzyme with its attached 23 amino acid signal peptide. The nucleotide sequence of the β-lactamase signal or hydrophobic leader sequence is illustrated by Fig. 5. The supernatant containing the β-lactamase enzyme was found to be approximately 80% pure by SDS

O PI gel analysis of proteins (Fig. 6, Lanes 4 and 5). Referring to Fig. 6, Lanes 1 and 5 are molecular weight standards. Lane 6 is the SDS gel analysis of proteins in the supernatant from E^ coli cells carrying pBR322 plasmid DNA. Lane 3 is the same for B^ subtilis BR151 cells carrying pC194 plasmid DNA. A finding that the β-lactamase enzyme disappears on DEAΞ cellulose and is not released from the column confirmed the presence of the β-lactamase signal sequence. This would be expected of a molecule retaining strong hydrophobic regions.

Cells of B^ subtilis strain BR151, following introduction of pLF119 plasmid DNA, were found to express both CAM and AMP resistance and produce a "clumpy" phenotype appearance, as illustrative in Fig. 4, in the absence of free plasmid or chromosomal integration of DNA fragments. This observation led tjo the discovery of a degradation product of the plasmid which has. been termed by the applicant as a quasi- plasmid or "quasmid" capable of replicating and expressing genes. In the pLF119 structure a B. subtilis replicative origin is inserted between the palindromic DNA sequences, with the result that a stem loop structure is formed when the plasmid is degraded (see Fig. 10). The electron microscope confirmation of this structure is shown in Fig. 8. The quasmid (1) replicates, (2) expresses genes, (3) is stably maintained in membrane complex in the bacterial cell, and (4) constitutes a novel method of maintaining and expressing foreign DNA in B^ subtilis.

Lysis of BR151 B^ subtilis cells carrying plasmid pLF119 produced no characteristic plasmid band following cesium chloride density gradient centrifugation. Probes of purified chromosomal DNA with CAT and AMP gene fragments from pC194 and pBR322 produced no evidence of chromosomal integration. Ampicillin and chloramphenicol resistance were not lost during curing with acridine orange in cells transformed with pLF119 DNA. The clumpy phenotype illustrated in Fig. 4 was produced in response to ampicillin pressure irrespective of the presence or absence of chloramphenicol. Transformation of BR151 with chromosomal DNA from BR151 expressing AMP, CAM, and the clumpy phenotype illustrated by Fig. 4 did not result in a transfer of any of these traits to the progeny. Plasmid curing experiments did not result in loss of the traits, nor did any plasmid obtained by standard techniques of isolation from the cells.

Experimental evidence confirming the presence of- the quasmid in B^ subtilis strain BR151 following introduction of pLF119 plasmid DNA was revealed by the above as well as characteristic weak plasmid-like bands obtained by prolonged SDS treatment of lysozyme lysed cells. Lysis by sonication revealed no evidence of these bands in the chromosomal or supernatant fraction; however, SDS treatment of the membrane or pellet fraction efficiently produced the same characteristic banding pattern. Similar bands were obtained from the bottom 3 cm of a cesium chloride gradient. These bands light up with both CAT and AMP probes. The DNA bands indicated by the arrow in Fig. 9 resisted RNAse and were sensitive to EXO III, indicating the presence of a free-double stranded end. In Fig. 9, Lane 1 is the molecular weight standard. Lane 2 shows the electrophoresis pattern of the DNA bands of B^ subtilis BR151 cells carrying pLF119 plasmid DNA-ampicillin amplified. Lane 3 shows the same for B^ subtilis BR151 cells carrying pC119 plasmid DNA without ampicillin amplification. Both S_]_ and single-stranded specific endonucleases degraded the bands; however, the bands were- resistant to many restriction endonucleases. Nicking the bands with DNAse did not alter the banding

position; however, a new band at 2.0 Kb on agarose was produced by this treatment. This same band was generated after Clal digestion of the quasmid. Anomalous migration of the bands which appeared at 1.1 and 10.3 Kb on agarose, but which banded at 25 Kb on 5% acrylamide, confirmed the presence of a degree of single-stranded character in the quasmid suggested by the Si and endonuclease sensitivity and the unusual banding position in cesium chloride. Weak binding of ethidium bromide also suggested this. The anomaly of the 1.1 Kb piece was particularly interesting, in that it bound CAT probe prepared from pC194 and also AMP probe prepared from pBR322. These probes were both well over 1.0 Kb and contained no homologous sequences. A structure consistent with the observed results is a hairpin loop with AMP regions of the DNA strand folded back into a double-stranded region, leaving a single-stranded loop consisting of the pC194 DNA insert region containing an active replicative origin (see Fig. 10). Electron microscopy studies illustrated by Fig. 8 support the observed results. In some cases, the looped region even showed short base pairing in regions .which would represent the pC194 replicon region and the inverted repeat region upstream from the chloramphenicol acetyl transferase gene. This structure, when nicked by DNAsel or cut with Clal produced a double stranded degradation product representing only the AMP region. This piece would normally migrate and bind ethidium bromide and be resistant to the single-stranded nucleases. Such characteristics are seen in the 2 Kb band which appears after Clal digestion of the quasmid, and also after DNAsel nicking. The 1.1 Kb band of Fig. 9 (indicated by the arrow) represents the quasmid of Fig. 8, which is defined by the LTR regions of the pLF119 plasmid. The 2 Kb band apparently represents a degradation artifact produced by nicking which removes the single- stranded loop.

Both the 1.1 Kb and the 10.0 Kb bands are produced by the host cell, whether by autonomous replication or degradation of other identified forms. The 10.0 Kb band appears to represent the whole double- stranded plasmid pLF119. Alternatively, it could be linear double-stranded concatameric DNA produced in some fashion by the 1.1 Kb band. The 10.0 Kb band is greatly enriched under AMP pressure, suggesting it is the source of the amplification of β-lactamase expression through increased copy number. Although the 1.1 Kb band remains unchanged quantitatively during ampicillin amplification, it apparently is important in the development of the amplified secreting system. Cells which lack a 1.1 Kb band are unable to undergo _f amplification of the 10 Kb band or increase expression of the gene product.

Introduction of the Cloning Vector into Host Cells

The cloning vector in the form of a chimeric plasmid, or transposable insertion sequence is introduced into the host cell by the known processes of genetic transformation, transfection, or . viral infection of the cells or protoplasts of the cell in a manner such that production and secretion to the cell surface of the product protein coded by the vector DNA ensues.

The host cells carrying- the cloning vector are isolated and cultured by conventional fermentation processes and produce a characteristic clumpy appearance when grown in liquid media, as illustrated by Fig. 4. The "clumped" cells are the result of accumulation of product protein synthesized in the transformed host cells and secreted to the exterior of the cell where it remains bound in large quantities to the surface of the cell. Alteration of the surrounding environment of the host cell by pH, temperature change, or ionic strength releases the entire uncleaved product protein and signal sequence from the cell surface, leaving the transformed cell available to produce an additional crop of protein product.

The cells may be periodically harvested and returned to grow and produce additional product in a continuous culture mode. The product protein released into the fluid medium may contain the signal sequence whose removal may require subsequent processing. Released product protein in the medium is present in active form at high concentration (see Fig. 6).

Levels of product protein production in the amplified system, calculated by quantitative assay of β-lactamase enzyme released from log growth cells, ranged consistently between 2 million and 20 million active enzyme molecules produced per cell over 4-6 hours of active cell growth—substantially more product in a more pure form than can be produced in E^ coli. The transformant cells may be immobilized on a support surface for culture and production of the product protein as described, for example, in Mosbach, et al.. Nature, 302 (7) 543-45 (1983).

The following examples are illustrative of the method and products of the subject invention, but are not to be construed as limiting. All percentages are by weight, and all solvent mixture proportions are by volume, unless otherwise specified. In the examples, the starting materials, buffers, and cell media are generally known. The cloning vehicles used were derived from the small plasmids pBR322 and pC194, both of which are well known.

The bacterial hosts used in the examples included strains of E coli HB101 (ATCC No. 33694) described by Boyer et al., J. Mol. Biol. 41, 459-472 (1969), and B^ Subtilis BR151 (ATCC No. 37705). The buffers and media used are commercially available and were purchased from Difco Corporation.

Example 1 Making the Cloning Vehicle from pBR322 and pC194

Referring to Fig. 1, DNA was isolated from pBR322 amplified in E. coli for 18 hours with chloram¬ phenicol. This DNA was further purified on a cesium chloride gradient. Analysis of the product on agarose and acrylamide gels indicated that it was plasmid DNA. Restriction fragments with AVA II matched those of pBR322. Restriction with Clal produced a single band of linear DNA representing the entire pBR322 sequence.

DNA from pC194 was isolated from B. subtilis and purified on cesium chloride. Restriction with Clal produced two bands of molecular weight, 1.7 Kb and 1.3 Kb, respectively. The 1.7 Kb fragment was cut from agarose gels and eluted by the "squeeze freeze" technique.

After extraction, the 1.7 Kb fragment of pC194 was ligated with the linear pBR322 fragments by standard procedures, except that the alkaline phos¬ phatase treatment was omitted in order to obtain the- inverted pBR322 dimer unit.

Following ligation, the construct was trans¬ formed into E. coli HB101 and grown on LB media with 25 μg/ml CAM. Transformants appeared after two days, and 32 clones were initially screened from single col¬ ony isolates. The recombinants were selected for abil¬ ity to grow in 25 μg/ml ampicillin and inability to grow in 25 μg/ml tetracycline. Restriction enzyme map- ping was carried out by conventional procedures to con¬ firm the structure of the plasmid illustrated in Fig. 2. The restriction endonuclease used included PST I, Hinds III, Ace I and Hinf I.

OMPI Example 2 Transformation of PLF119 into B. Subtilis Strain BR151

B. subtilis strain BR151 was grown in HS media for 4-1/2 hours and transferred into LS media at 1:5 dilution. Cells were grown in LS media for 90 min¬ utes. 0.2 μg of DNA of pLF119 extracted from E. coli, HB101, were added to 1 mL of competent cells and incu¬ bated for 30 minutes at 37°C. The reaction was termi- nated by adding 2.5 μg of DNase dissolved in 0.15 M sodium chloride. Cells were incubated for 2 hours .at 37°C. for expression or drug resistance. Clones were plated on CAM/AMP fortified. TBAB plates for selection. Sixteen clones were selected from competent cell trans- formation and streaked on CAM/AMP TBAB plates. These clones were grown in 10 ml . broth culture containing 25 μg/ml CAM/AMP. The clone appearing to have the "clumpiest" growth chracteristic was chosen for β-lac¬ tamase assay. A culture of the chosen transfor ant was grown 2 hours in 10 ml PA plus 25 μg/ml CAM/AMP. Cells were pelleted and resuspended in l/20th volume of STE buffer. Part of the resuspended cells were heat treated at 55°C. for 15 minutes while the other half were left at room temperature. All cells were again pelleted and the supernatant fluid assayed for β-lac¬ tamase activity. The assays were run using nitrocefin eluted from nitrocefin impregnated antibiotic testing discs (Cefinase, BBL Microbiological) by soaking in buffer for one hour to release the nitrocefin. Nitro- cefin, elution 0.9 ml, was placed in a cuvette with 0.1 ml supernatant from the cultures. Optical densi¬ ties were taken at . 2-1/2 minute intervals for 30 minutes at 510 nm. Control cuvettes contained solu¬ tions of commercially purified β-lactamase enzyme in 0.9" ml nitrocefin. The results are shown in Fig. 7.

-TξxTZE Example 3

Culturing Bacterial Hosts Transformed with Cloning Vehicles

pLF119, derived from E. coli HB101, was transformed as previously described into B. subtilis strain BR 151. The transformants were screened for acquisition of resistance to 25 μg/ml AMP and CAM. Clones which satisfied the antibiotic resistance cri- teria were grown in PA broth under ampicillin pressure and visually screened for production of pronounced "clumpy" phenotype as shown in Fig. 4. Cultures of B. subtilis strain . BR151 carrying the introduced pLF119 plasmid which had been grown on CAM/AMP containing TBAB plates were streaked onto TBAB plates containing CAM/AMP, CAM, AMP, and a plate with no antibiotics and the plates incubated overnight at 37°C. Cells were removed from each plate and used to inoculate 40 ml of a PA broth containing CAM/AMP, CAM, AMP, and no anti- biotics. Again, cells were taken from each plate and centrifuged at 4000 rpm for 5 minutes. The supernatant was removed and the cells resuspended in 5 ml STE buffer and incubated at 50°C. for 15 minutes. The cells were pelleted and the supernatant saved to test for β-lactamase activity. The pellet was redissolved in 500 μg STE buffer and 500 μg/ml lysozome and incu¬ bated for 30 minutes at 37°C. Fifty μl 10 percent SDS, 50 μl 250 mM EDTA were added, and the admixture incubated for another 30 minutes at 50°C. The solution was extracted 2 X with chloroform and 1 X with phenol:chloroform (50/50) and again with chloroform. The DNA was then precipitated with 3 vol. of ethyl alcohol for 1 hour at 70°C. and centrifuged at 10K rpm for 20 minutes. The pellet was redissolved in 100 μl TE buffer and 200 μg/ml RNase and incubated at 37°C. for 30 minutes. The RNase reaction was carried out in a monium acetate and precipitated by addition of three volumes ethyl alcohol. The DNA was collected by cen- trifugation at 10K rpm for 20 minutes, resuspended in 2 μl ammonium acetate, and reprecipitated with three volumes of ethyl alcohol. The DNA pellet was redissolved in 100 μl TE buffer and stored at -20°C. Protein extracts from each culture were assayed for β- lactamase activity. One hundred μl of each sample was mixed with 900 μl nitrocefin solution in 0.1 molar ammonium acetate and read at various times by spectro- phometric assay techniques. Controls of B. subtilis strain BR151, containing plasmid pC194 and BR151 containing plasmid pWRlOl were also run with a β- lactamase standard. The results are as shown in the graph of Fig. 7.

OMPI

Claims

Claims

1. A method of concentrating and purifying secreted product proteins produced by a cloning vector present in a transformant cell, comprising; providing transformant cells carrying a clon¬ ing vector which results in expression of a product protein affixed to or as a part of a signal sequence cloned adjacent to the structural gene coding for the product protein, the signal sequence derived from a source different than the transformant cell being used to produce the product protein, the non-host signal sequence mediating transport of the product protein across the membranes of the transformant cells with the product protein remaining bound to the surface of the transformant cells where it concentrates in large quantities; culturing the transformant cells in a fluid medium under appropriate nutrient conditions conducive to expression of the product protein; and altering the environmental conditions sur¬ rounding the transformant cells to release the surface- bound product protein.

2. The method of claim 1, including, prior to altering the environmental conditions surrounding the transformant cells, concentrating the transformant cells with the product protein retained bound to the cell wall in a small amount of fluid medium.

3. The method of claim 2 wherein the environmental conditions are altered such that the product protein is released into the fluid medium in a biologically active form.

4. The method of claim 2 wherein the product protein is released into the fluid medium as an inactive precursor to a biologically active form which may be activated by subsequent processing.

GMPI

5. The method of claim 2, including return¬ ing the transformant cells to active growth following release of the surface-bound product protein to produce a subsequent crop of product protein.

6. The method of claim 2 wherein the non- host signal sequence is not cleaved from the product protein following transport of the product protein across the membrane of the transformant cells because of the absence of enzymes in the transformant cells to effect such cleavage, thereby allowing the uncleaved signal sequence/product protein to accumulate at the surface of the transformant cells.

7. The method of claim 6 wherein the non- host signal sequence is an integral part of the cloning vector.

8. The method of claim 1 wherein the step of altering the environmental conditions surrounding the _ transformant cells is by adjustment of temperature, pH, or ionic strength of the fluid media.

9. The method of claim 1 wherein the trans¬ formant cells producing the product protein are immobilized upon a support surface for culture and production of the product protein.

10. The method of claim 1 including introduc- ing the cloning vector into the transformant cell by genetic transformation, transfection, or viral infec¬ tion of the transformant cells or protoplasts of the transformant cells in a manner such that production and secretion of the product protein to the cell surface ensues.

11. The method of claim 1 wherein the cloning vector is in the form of a plasmid carrying the DNA coding for the product protein and the signal sequence.

12. The method of claim 11 wherein the clon- ing vector is in the form of a chimeric plasmid carry¬ ing DNA coding for the product protein and the non-host

" signal sequence, the chimeric plasmid capable of repli¬ cating in the transformant cells to express the product protein.

13. The method of claim 10 wherein the clon- ing vector is a sequence contained within a transposable insertion sequence.

14. The method of claim 1 wherein the trans¬ formant cells are cells having cell-wall properties common to bacterial cells classified as "gram- positive".

15. The method of claim 14 wherein the trans¬ formant cells _,are the gram-positive bacterium, Bacillus subtilis.

16. The method of claim 10 wherein the clon- ing vector is comprised of inverted DNA sequences of the plasmid pBR322 spanned by a 1.7 Kb fragment of plasmid pC.194.

17. The method of claim 11 wherein the clon¬ ing vector contained in the transformant cells is characterized by the restriction map of Figure 2 of the drawings.

18. The method of claim 16 wherein the clon¬ ing vector contained in the transformant cells is plas¬ mid pLF119 having ATCC Deposit Accession No. 39380 introduced into B. subtilis, the DNA carried by the plasmid actively expressing product proteins coded for by the ampicillin region of the E. coli-derived pBR322 moiety in the chimera, the clones having the ability to express E. coli foreign protein gene in the B. subtilis host cell.

19. The method of claim 18 wherein the expressed product protein is the β-lactamase enzyme coded by the pBR322 DNA AMP gene region.

20. A transformant cell containing a stable, foreign, cloning vector capable of expressing a product protein affixed to or as part of a non-host signal

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> sequence cloned adjacent to the structural gene for the product protein, the non-host signal sequence mediating transport of the product protein across the membrane of the transformant cell, with the product protein re ain- ing bound to the surface of the transformant cell where it concentrates in large quantity.

21. The transformant cell of claim 20 wherein the cell is gram-positive.

22. The transformant cell of claim 20 wherein the non-host signal sequence is derived from a gram- negative cell.

23. The transformant cell of claim 20 wherein the cloning vector is introduced into the cell by genetic transformation, transfection, or viral infection of the cell or protoplasts of the cell.

24. The transformant cell of claim 20 wherein the cloning vector is in the form of a plasmid carrying the DNA coding for the product protein and the signal sequence.

25. The transformant cell of claim 20 wherein the cloning vector is in the form of a chimeric plasmid carrying DNA coding for the product protein and the non-host signal sequence, the chimeric plasmid capable of replicating in the transformant cell and expressing the product protein.

26. The transformant cell of claim 20 wherein the cloning vector is a vector sequence contained within a transposable insertion sequence.

27. The transformant cell of claim 25 wherein the host cell is the gram-positive bacterium, Bacillus subtilis.

28. The transformant cell of claim 21 wherein the chimeric plasmid introduced into the transformant cell - is comprised of inverted sequences of the plasmid pBR322 spanned by a 1.7 Kb fragment of plasmid pC194.

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29. The transformant cell of claim 20 wherein the chimeric plasmid introduced into the transformant cell is characterized by the map in Figure 2 of the drawings.

30. The transformant cell of claim 20 wherein the cloning vector contained in the transformant cell is plasmid pLF119 having ATCC Deposit Accession No. 39380 introduced into B. subtilis, the DNA carried by the plasmid actively expressing product proteins coded for by the ampicillin region of the E. coli- derived pBR322 moiety in the chimera, the clones having the ability to express the E. coli foreign protein gene in B. subtilis.

31. The transformant cell of claim 30 wherein the expressed product protein is the β-lactamase enzyme coded by the pBR322 DNA AMP gene region.

32. The tranformant cell of claim 30 wherein the product β-lactamase enzyme protein is released from cells by mild heating and displays full enzymatic activity.

33. A cloning vector adapted for transforma¬ tion of a microbial host, comprising an active replica¬ tion origin site including a structural gene positioned between complementary strands of adjacent inverted repeated DNA sequences which form base pairs with each other, the cloning vector capable of replicating and expressing genes.

34. The cloning vector of claim 33 wherein the complementary strands of adjacent inverted repeated DNA sequences are ampicillin coding sequences or struc¬ tural analogs thereof which have no antibiotic activity.

35. The cloning vector of claim 33 wherein the active replication origin site is derived from the Staph. aureus plasmid pC194 and the nverted repeated DNA sequences are derived from the E. coli plasmid pBR322.

36. The cloning vector of claim 33 character¬ ized by the restriction map of Fig. 2 of the drawings.

37. The cloning vector of claim 33 known as plasmid pLF119 in B. subtilis having ATCC Deposit Accession No. 39380.

38. A method of inducing overexpression of product protein produced by a cloning vector present and secreted by the transformant cell, comprising: providing transformant cells carrying a clon- ing vector having an active replication origin site including a structural gene positioned between comple¬ mentary strands of adjacent inverted repeated DNA sequences which form base pairs with each other; and culturing the transformant cells in a fluid media under appropriate nutrient conditions conducive to expression of the product protein.

39. The method of claim 38 wherein the complementary strands of adjacent inverted DNA sequences are selected from the group consisting of ampicillin coding sequences and structural analogs thereof which have no antibiotic activity.

40. The method of claim 38 wherein the active replication origin site is derived from the Staph. aureus plasmid pC194 and the inverted repeated DNA sequences are derived from the E. coli plasmid pBR322.

41. The method of claim 38 wherein the clon¬ ing vector is characterized by the restriction map of Fig. 2 of the drawings.

42. The method of claim 38 wherein the clon- ing vector is plasmid pLFH9 in B. subtilis having ATCC

Deposit Accession No. 39380.

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