WO1995010620A1

WO1995010620A1 - A plasmid vector useful for the expression of a foreign dna sequence

Info

Publication number: WO1995010620A1
Application number: PCT/US1994/011719
Authority: WO
Inventors: Richard M. Synenki; James R. Mcmullen; Christopher A. Zook
Original assignee: Mallinckrodt Veterinary, Inc.
Priority date: 1993-10-15
Filing date: 1994-10-14
Publication date: 1995-04-20
Also published as: ZA948096B; EP0723594A1; AU7980394A

Abstract

The present invention relates to expression vectors which permit the easy insertion of any desired DNA sequence into the vector, to recombinant organisms transformed with those vectors and to polypeptides produced by the transformed organisms. The vector includes a promoter, a transcription initiation site, a sequence of DNA encoding a portion of the N-terminus of the protein Δ7-pST, at least a first synthetic oligonucleotide linker, a transcription terminator and a selective marker, in that order and operatively linked to one another.

Description

A PLASMID VECTOR USEFUL FOR THE EXPRESSION OF A FOREIGN DNA SEQUENCE

Backcrround of the Invention This invention relates to compositions and methods useful for producing proteins in recombinant hosts, particularly in microbial hosts. The invention is especially directed to the production of fusion proteins.

The invention employs recombinant DNA technology which is directed to the efficient expression of cloned DNA by a recombinant host. It is considered desirable to obtain the expression product, the protein, in as high a yield as possible. Such proteins can have numerous uses, including, for example, as agents for the treatment or diagnosis of various pathologies such as diseases or infections. It is well established that either procaryotic or eukaryotic proteins can be expressed in recombinant hosts where such proteins are not normally present in those hosts. Generally, these techniques are carried out by inserting the DNA sequence which codes for the proteins of interest downstream from a control region containing a promoter/operator and ribosome binding site located in plasmid DNA. The plasmid containing the DNA to be expressed is inserted into a cell to transform the cell which then can be cultivated to produce or express the protein of interest.

As used herein, "heterologous protein" means a protein which is not naturally produced by the host cell . Used interchangeably with heterologous protein throughout the specification are terms such as "foreign" protein and protein of interest. Similarly, foreign gene, cDNA, and gene or DNA of interest each refer to the DNA sequence encoding the heterologous protein.

While expression of a foreign protein, conceptually, is a straightforward procedure, in practice there are a number of obstacles to be overcome prior to getting a cell to synthesize such a protein. One such obstacle can occur during transcription of the DNA. During the process of transcription the messenger RNA can develop secondary structure, i.e., loops, which will inhibit the passage of ribosomes. If ribosomes cannot pass freely along the gene, expression of the gene will be inhibited and the resultant protein will not be produced. It is an object of the present invention to overcome this problem. In the past, cloned DNA has been expressed as a fusion protein to take advantage of prokaryotic control sequences. The protein encoded by the DNA of interest is expressed as the C-terminal end of the fusion protein following the construction of an expression vector in which the DNA encoding the protein of interest is operably linked to, and inserted downstream from, DNA encoding a prokaryotic polypeptide. For example, U.S. Patent 4,820,642 (Edman, et al . ) teaches the use of restriction enzymes and the construction of fusion proteins. In using these prior art procedures a problem could arise with the placement of the DNA sequence of interest into the correct reading frame phase within the plasmid. Modification of the sequence length by the addition of one or two nucleotides usually was necessary. An additional problem with fusion proteins generated according to the teaching of the prior art is that many contained large numbers of amino acids from the prokaryotic sequence. It is one object of the present invention to overcome these problems.

Henceforth, as used herein, "gene" shall include cDNA sequences as well as naturally isolated DNA and synthetic DNA encoding polypeptides.

Summary of the Invention

The present invention provides compositions and methods for the easy cloning of a wide variety of genes or cDNA sequences and high expression of those genes under the control of promoters such as the bacteriophage λ heat inducible P_L promoter. Any gene or sequence of cDNA can be cloned into an appropriate reading frame phase without sequence modification. The plasmid vector of this invention can be referred to as a universal cloning vector, because the vector without modification can be used to express a wide variety of foreign genes and will result in high expression of the desired protein product. In a preferred embodiment, the plasmid comprises the following elements, operably linked to one another in the following sequence: a P_L promoter, a ribosome binding site, the first 30 base pairs of the Δ7-pST gene, a 21 base pair first ("front- end") synthetic oligonucleotide containing multiple blunt end restriction enzyme sites, a second ("back- end") synthetic oligonucleotide containing at least one restriction enzyme site, a transcription terminator and a drug resistance marker. Brief Description of the Drawings Figure 1 is a schematic illustration of a preferred embodiment of the present invention.

Figure 2 describes a preferred embodiment of a front-end oligonucleotide containing multiple blunt end restriction enzyme sites. (SEQ. ID. NOS:1 AND 2) .

Figure 3 sets forth the sequence of a preferred embodiment of a back-end oligonucleotide. (SEQ. ID. NOS:3 AND 4) . Figure 4 sets forth a preferred transcription terminator dimer sequence. (SEQ. ID. NOS:5 AND 6) .

Figure 5 is a schematic diagram showing a preferred plasmid of the present invention with the restriction enzyme sites contained therein. Figure 6 depicts plasmid pLA107 described herein. Figure 7 depicts plasmid pCZOOl described herein. Figure 8 depicts plasmid pCZ301 described herein.

Detailed Disclosure of the Best Mode of Practicing the Invention The subject invention relates to the expression of a foreign DNA sequence in a host organism by a plasmid vector. More particularly, the invention is directed to a plasmid vector and its use for high-efficiency expression of desired proteins. In general, as illustrated in Figure 1, which shows a schematic of a preferred embodiment, the plasmid vector will contain, in an operatively linked manner, DNA encoding a promoter 1, a ribosome binding site 2, DNA encoding a portion of the N-terminus of the Δ7 porcine somatotropin ("pST") protein 3, a first synthetic oligonucleotide 4, a second synthetic oligonucleotide 5, a transcription terminator 6, and a selection marker 7. It is to be understood that this schematic is not representative of the relative sizes of these various elements.

Operatively linked upstream from the ribosome binding site is a promoter site. Promoters are well- known to those of skill in the art. The promoter provides effective control of the expression process. The invention contemplates the use of any promoter. However, an especially preferred promoter is the lambda phage P_L promoter described, for example, in published European application EP0104920. This promoter is well- known to those of skill in the art and provides for temperature control of expression.

Operatively linked at the 3' end of the promoter DNA is a ribosome binding site, alternatively termed a transcription initiation site, comprising the DNA sequence ATG. An especially preferred ribosome binding site is from the bacteriophage Mu.

Downstream from the transcription initiation site is operatively linked DNA encoding a portion of the N- terminus of the protein Δ7-pST. Δ7 porcine somatotropin is a protein comprising full length porcine somatotropin minus the first seven amino acids at its N-terminus. Expression of the DNA sequence encoding Δ7-pST is disclosed in EP0104920. While the number of base pairs encoding the portion of the N- terminus of Δ7-pST used in the vectors of this invention can vary, a preferred range is from about 21 to about 60 base pairs. Those of skill in the art will recognize that this range of base pairs corresponds to the first 7-20 amino acids of the N-terminus. An especially preferred range is from about 30 to about 45 base pairs. Most preferred is a segment comprising 30 base pairs and encoding the first 10 amino acids of the N-terminus of Δ7-pST. Use of the portion of the N-terminus of Δ7-pST DNA as described above promotes the efficient expression of this fusion protein. Without wishing to be bound by theory, it is believed that the DNA encoding a portion of the N-terminus of Δ7-pST minimizes secondary structure in the messenger RNA produced during transcription. The minimization of secondary structure increases the rate of translation.

The scope of this invention encompasses the use of DNA sequences which encode analogs of the N-terminal 7- 20 amino acids of Δ7-pST. For example, nucleotides encoding a single amino acid within that N-terminal sequence can be deleted or substituted by nucleotides which encode a different amino acid. Modifications to the Δ7-pST DNA sequence which would not hinder the desired efficient expression of the desired fusion protein can be made by persons of skill in the art with routine experimentation. In the preferred embodiments of this invention, the DNA sequence encodes the N- terminal 7-20 amino acids of Δ7-pST.

Operatively linked to the 3' end of the DNA encoding a portion of the N-terminus of Δ7-pST is a first synthetic oligonucleotide. This oligonucleotide can be synthesized by known techniques as a single strand and annealed with its complementary strand to form a double stranded DNA sequence comprising at least one blunt end restriction enzyme site. Preferably, this linker contains 3 unique blunt-end restriction enzyme sites, each of which can be cut to provide an opening within the vector at a different phase within the reading frame established by the translation initiation site and the DNA sequence encoding the N- terminus of Δ7-pST. The presence of three different unique blunt end restriction enzyme sites permits selection of an appropriate restriction enzyme to cleave the DNA such that foreign DNA can be ligated into the resultant insertion site in the correct reading phase with the Δ7-pST DNA as previously described without additional modification. Blunt end restriction enzymes are known to those of skill in the art. The DNA encoding any blunt end restriction enzyme site can be used. The DNA sequence of a most preferred first synthetic oligonucleotide is shown in Figure 2. In one embodiment, only a single synthetic oligonucleotide is used. In this embodiment, after cleaving the oligonucleotide with a blunt-end restriction enzyme, a foreign gene can be inserted directly into the opened vector. The N- and C- terminals of the foreign gene need only be blunt-ended using known techniques prior to ligation to the blunt ends of the opened vector. This embodiment requires that the orientation of the foreign gene be determined after insertion. Techniques for blunt-ending DNA are known to those of skill in the art, as are techniques for ligating DNA together and determining the orientation of inserted DNA..

In a second embodiment, the plasmid will contain a first and a second synthetic oligonucleotide. The first is linked to the 3' end of the DNA sequence encoding the N-terminus of Δ7-pST as described above and, in this embodiment, can be referred to as a "front-end" oligonucleotide. The second oligonucleotide can be referred to as a "back-end" oligonucleotide. The second oligonucleotide can be inserted into the expression vector by operatively linking its 5' end downstream of the 3' end of the first oligonucleotide.

The back-end oligonucleotide contains at least one restriction enzyme site. Preferably, the oligonucleotide will contain multiple unique restriction enzyme sites, each of which result in overhanging, or sticky, ends when cut with a restriction enzyme. Of course, the second oligonucleotide also can contain blunt-end restriction enzyme sites. However, the use of a back-end oligonucleotide which, upon cutting with a restriction enzyme, results in a sticky end is preferred because the DNA of interest can easily be ligated thereto. Sticky end ligations are easier to perform in the laboratory as a practical matter, as is known to those of skill in the art. An especially preferred back-end oligonucleotide sequence is illustrated in Figure 3. Additionally, the combined use of a blunt-end site in the front-end oligonucleotide and a sticky-end site in the back-end oligonucleotide ensures that the DNA of interest can be modified such that when it is inserted into the vector, it will be in the correct orientation. The vector is treated with two restriction enzymes which recognize restriction enzyme sites within the first oligonucleotide linker and within the second oligonucleotide linker, respectively, resulting in a linearized vector having one blunt end and one sticky end and a small excised piece of DNA which also has one blunt end and one sticky end. The foreign DNA is ligated into the opened vector such that the 5' end of the foreign DNA is ligated to the blunt-end and the 3' end is ligated to the sticky end of the vector. Alternatively, the front end oligonucleotide can be selected such that it contains three unique restriction sites, each in a different reading phase, and each of which, when cut with the restriction enzyme which recognizes the site, will produce a sticky end. The back end oligonucleotide can be selected such that it can be cut with a restriction enzyme so as to produce a blunt end.

In a third embodiment of the present invention, a single oligonucleotide combining the features of the above-described front- and back-end oligonucleotides is operatively annealed to the 3' end of the DNA encoding a portion of the N-terminus of Δ7-pST. In this embodiment, the oligonucleotide preferably will contain both blunt and sticky end restriction enzyme recognition sites. Typically, the sticky end sites are downstream of the blunt-end sites. Foreign DNA can be inserted into this oligonucleotide as previously described with respect to the second embodiment. It is to be understood that the present invention encompasses the use of synthetic oligonucleotides, whether single, front-end or back-end, of any effective length. Thus, as many, or as few, restriction enzyme sites as desired can be included. Any of a wide variety of foreign genes can be inserted into the expression vector of the present invention. The gene need only be isolated using techniques known to those of skill in the art. Typically, this involves digesting with appropriate restriction enzymes at both the 5' and 3' ends of the gene. Synthetic genes also can be used.

If the heterologous gene to be used was isolated using a blunt-end restriction enzyme digest of its 5' end during the isolation process, it can be directly ligated to the blunt end of the first oligonucleotide linker produced by blunt end digestion of that oligonucleotide. If the 5' end of the heterologous gene was subjected to a sticky-end digestion, it can be filled in, i.e., blunt ended, using various DNA polymerase reactions known to those of skill in the art prior to ligation of the 5' end to the blunt-end cut in the synthetic oligonucleotide.

The same ligation procedures as described above can be applied to the 3' end of the gene, if the gene is to be ligated into a single blunt-end site in the oligonucleotide.

Where the linearized vector has one blunt end and one sticky end, the above-described techniques can be used for ligation of the 5' end of the gene. Ligation of the 3' end of the gene to the sticky site on the synthetic oligonucleotide proceeds according to techniques known to those of skill in the art.

Operatively linked immediately downstream from the sole, or back-end, oligonucleotide linker is a transcription terminator. Transcription terminators are known to those of skill in the art and cause the RNA polymerase to dissociate from the DNA. Typically, a transcription terminator can form a hair-pin loop. Its presence insures that mRNA is made from only relevant regions of DNA. While any transcription terminator can be used, the sequence of a preferred terminator is illustrated in Figure 4.

Operatively linked downstream from the transcription terminator can be a DNA sequence encoding a selective marker, including, without limitation, an ampicillin, tetracycline, kanamycin, etc. resistance marker. Such markers are known to those of skill in the art and are useful in selecting the plasmid vectors of the present invention.

The invention having been generally described, the following non-limiting examples are provided to further illustrate the invention. Example 1. Construction of the Plasmid pCZOOl

To construct the cloning vector, plasmid pLA107 was restriction enzyme digested. Plasmid pLA107 is illustrated in Figure 6. This plasmid contains two Apa I restriction enzyme sites. Cleavage with the restriction enzyme Apa I produced a 212 base pair Apa I/Apa I fragment which can be removed. Additionally, pLA107 contains, operatively linked, the P_L promoter, a Mu ribosome binding site, and a DNA sequence encoding the first 10 amino acids of the N-terminus of Δ7-pST. If other plasmids are selected, it is within the skill of those in the art, given the present disclosure, to ensure that a control region and a selected portion of DNA encoding a portion of the N-terminus of Δ7-pST are present in the plasmid.

After complete digestion, the plasmid was isolated and phosphatased using calf intestinal alkaline phosphatase ("CIP") . Such techniques are known to those of skill in the art. A front-end oligonucleotide (here shown in Figure 2) was ligated to the linearized, phosphatased plasmid, pLA107. The oligonucleotide can be synthesized by any means known to those of skill in the art. Various commercial organizations can synthesize DNA strands on demand as specified. Typically, oligonucleotides are single stranded DNAs and in the present invention the DNA is annealed with its complement to form a double stranded DNA.

In a preferred embodiment, the oligonucleotide containing a restriction enzyme recognition site which, when cut by the restriction enzyme, results in two blunt ends (Figure 2) was synthesized at the University of Illinois Biotechnology Center. This oligonucleotide contains the recognition sites EcoR V, Sma I and Pvu II. The synthesized oligonucleotide was shipped as two single stranded homologous DNAs. These single stranded DNAs were annealed to form a double stranded DNA and ligated to the Apa I-digested and phosphatased pLA107 plasmid. The ligated DNA was transformed into C600X+ Escherichia coli (E. coli) and plated on LB agar with ampicillin. Plasmid DNA was isolated from selected transformants and assayed for the oligonucleotide insertion, and its orientation was determined by restriction enzyme digestion analysis.

Plasmids were first screened for an EcoR V restriction enzyme site. A plasmid which is linearized by the action of EcoR V must contain the oligonucleotide insert since the restriction enzyme site is unique to the oligonucleotide and the new plasmid construction. The orientation of the insert was determined by partial digestion with Sma I; the large fragment was isolated and religated. Removal of the smallest Sma I fragment when the oligonucleotide is inserted in the correct orientation removes the unique EcoR V restriction enzyme site. If the oligonucleotide is inserted in the reverse orientation, the EcoR V site would remain in the ligated plasmid. The ligated plasmid DNA was digested with EcoR V. Plasmids which were no longer digested with the EcoR V enzyme contained the oligonucleotide in the proper orientation.

Alternatively, the orientation could be determined using the Pvu II site located in the front-end oligonucleotide. In this instance, upon religating the large fragment, the religated DNA would maintain the EcoR V site if the oligonucleotide was inserted in the correct orientation. The EcoR V site would no longer be present if the synthetic oligonucleotide had been inserted in the reverse orientation.

It is to be understood that the above described principles for determining presence and orientation of the front-end oligonucleotide can be applied using other restriction enzymes and plasmids. Such determinations, given the present teaching, are within the ambit of those of skill in the art.

In the present instance, subsequent DNA sequencing of the plasmid constructed showed that two oligonucleotides had been inserted into the linearized pLA107. The di er consisted of one oligonucleotide in the desired orientation ligated to a second oligonucleotide in the reverse orientation. This plasmid was named pCZOOl and is illustrated by Figure 7. Plasmid pCZOOl was isolated using the Cs-Cl EtBr cleared lysate plasmid isolation technique as described in Sambrook, J., E.F. Fritsch, T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd Edition, incorporated herein by reference. The extra front end oligonucleotide subsequently was excised, as described in example 4, below.

Example 2. Preparation of pCZ301

In order to construct an expression vector containing an additional, "back-end" oligonucleotide (illustrated in Figure 3) , plasmid pCZOOl from Example 1 was double-digested with restriction enzymes Hind III and Xba I. This digestion excised a 48 base pair portion from the plasmid. The remaining large fragment DNA was isolated from the 48 base pair Hind III/Xba I fragment and phosphatased using CIP as previously discussed. The back-end oligonucleotide then was ligated into the digested phosphatased large fragment of plasmid DNA. The ligated plasmid DNA containing the back-end oligonucleotide was transformed into C600X+ E. coli and selected for ampicillin resistance. To determine whether the back-end oligonucleotide was present and in the correct orientation, the plasmid was subjected to multiple restriction enzyme analysis. The plasmid DNA was digested with the Kpn I restriction enzyme. This restriction enzyme site is unique to the back-end oligonucleotide. The plasmid was not checked for orientation of the insert in this instance because it was only possible for the oligonucleotide to be inserted in one direction. After locating those plasmids containing the presence of the oligonucleotide they were isolated by the CsCl-EtBr cleared lysate plasmid DNA isolation technique and named pCZ301, as illustrated by Figure 8.

Example 3. Preparation of pCZ305

The last component of the universal cloning vector is a transcription terminator. Plasmid pCZ301 was digested with Hind III and assayed for complete digestion. The completely digested plasmid was isolated and phosphatased. A 330 base pair transcription terminator dimer referred to as Rao33 illustrated in Figure 4 was ligated into the digested and phosphatased pCZ301. Again, the ligated DNA was transformed into C600λ+ E. coli and plated on agar plates containing ampicillin. The selected transformants were cultured and the plasmid DNA isolated to ensure that the terminator was present. The plasmid was named pCZ305. The presence of the 330 base pair terminator insert was detected by digestion with Kpn I. This digestion, if the terminator is present, produces a visually detectable fragment on a one percent agarose gel. To ensure correct orientation, restriction enzyme digestion with Kpn I and Sal can be used. The double digest will cut a fragment of approximately 340 base pairs when in the proper orientation. This base pair fragment is of sufficient size to be visualized by 10 percent acrylamide gel electrophoresis. Had the terminator been inserted in the reverse orientation, the double digestion fragment would be too small to visualize using 10% acrylamide gel electrophoresis.

Example 4. Construction of pCZ306

The pCZ305 plasmid was digested with Pvu II restriction enzyme, the large fragment isolated and ligated, removing a 323bp fragment. The ligated DNA was transformed into C600X + E. coli and plated on LB agar with plasmid DNA isolated.

The plasmid DNA was assayed for the proper construction by restriction enzyme analysis with Apa I and Kpn I. Plasmid of the correct construction would be approximately 325bp smaller than plasmid pCZ305 when digested with Kpn I. A double digest would show a single band at approximately 285bp on a 10% acrylamide gel .

Plasmids containing the correct construction were purified and prepared by CsCl-EtBr cleared lysate plasmid isolation techniques, named pCZ306 and deposited with the American Type Culture Collection ("ATCC"), Bethesda, Maryland and given accession number 75500.

Example 5. Expression of Fusion Proteins in pCZ306 pCZ306 can be used to express DNA encoding a variety of desired proteins, including -glucuronidase, glucose isomerase, amylases, rennet, esterases, and oxygenases, e.g., lignin degrading enzymes and aromatic ring-cleaving enzymes. Other useful proteins which can be expressed with this system include hormones such as insulin, interferons, interleukins, somatotropins, somatostatins, somatomedins, growth factors, EGF, tumor necrosis factors, glucagon, hypothalamic hormones such as growth hormone stimulating factor, ACTH, endorphin, and adrenal derived protein hormones. This invention also can be used in the expression of bacterial toxins and viral proteins.

Preferably, however, the invention is used to produce EGF or porcine somatotropin. Until the present invention, it was difficult to recombinantly produce the native form, or complete N-terminus form, of porcine somatotropin. Additionally, the present invention can be used to produce modified somatotropins such as those disclosed in U.S. Patent Application No.

07/553,511 or EP0355460A2, both of which are incorporated herein by reference. A. Preparation of full length porcine somatotropin (N-terminal completed pST)

A gene coding for the entire porcine somatotropin was inserted into the plasmid pCZ306 of the present invention as described below and illustrated in Figure 5. The porcine somatotropin gene was obtained from plasmid pFN300. The somatotropin gene in this plasmid was constructed by ligating a synthetic oligonucleotide that coded for the first seven amino acids of the pST gene to the delta-7pST gene from plasmid pIClOl (available from the American Type Culture Collection, ATCC Accession No. 53031) .

Plasmid pFN300 was digested with Pvu II and BamH I restriction enzymes and the 3160 base pair (bp) fragment purified from an agarose gel. Plasmid FN300 was digested with the restriction enzyme Nco I . The resultant digest then was treated with T4 polymerase to fill in the four base pair 5' Nco I overhang. This enabled the N-terminus of the pST gene to be blunt-end ligated into the Pvu II site of the pCZ306 plasmid. The ligated product was restriction enzyme digested with BamH I and the resulting 670 b.p. fragment purified from an agarose gel . The 3160bp and 670bp DNA fragments which had been isolated as described were ligated, transformed into C600λ+ and plated on selective media. The correct plasmid construction was verified by restriction enzyme digestion and DNA sequence analysis. This plasmid was named pCZ308 and it has been deposited with the ATCC and given Accession Number 75502.

To evaluate the expression capabilities of pCZ308 a comparative test was carried out as described below. pFN300 is a plasmid containing the N-terminal complete porcine somatotropin. Plasmid pCZ308, as noted above, comprises the gene for N-terminal complete pST downstream from DNA encoding the N-terminal of Δ7-pST. The pIClOl plasmid available from ATCC Accession No. 53031 contains the Δ7-pST gene. This plasmid was used as a control. All of the plasmids were transformed into and expressed in E. coli HB101 under the control of a P_L promoter. Also present in each of the cells was plasmid pcI857 which contains the temperature sensitive repressor cI857. The seed medium for the expression experiment was ESM-2 medium (described in Table I) plus ampicillin (100 mg/ml) and kanamycin (50 mg/ml) . The seeds consisted of 100 ml of the medium in a 500 ml Erlenmeyer flask. The flasks were inoculated with 0.1 ml of frozen culture (E. coli containing plasmid pCZ308, pFN300 or pIClOl) from vials stored at -80°C and incubated for 16 hours at 30°C on a rotary shaker at 350 RPM. Filter sterilized thiamine, about 0.2 parts per million, was added just prior to inoculation. Duplicate flasks were inoculated with 1.25 ml of the respective seed cultures and incubated for 1 hour at 30°C on a rotary shaker at 350 RPM. The temperature of the incubated flasks was raised to 40°C for six hours to induce the production of the desired somatotropin.

Flasks were assayed for growth and the production of protein by measuring optical density at 550 nanometers. Table II shows the average optical density obtained for cells containing plasmids pFN300, pCZ308 and pIClOl. Duplicate flasks were combined and a sample was analyzed by HPLC. The results of that analysis also appear in Table II.

Table I

ESM-2 Medium

NZ Amine A 23 g

Glycerol 30 g

(NH₄)₂ S0₄ 5 g

K₂HP0₄ 6 g

NaH₂P0₄ 3 g

NaCitrate 1 g

MgS0₄ 3.s g

Biogen T. E. Sol'n 20 ml

Distilled H₂0 to 1000 ml pH to 6.8 with NaOH Sterilized 15 min at 121 C

Add sterile-filtered Ampicillin (100 ug/ml) kanamycin (50 ug/ml) before inoculation.

Table II

Plasmid Seed OD Final OD Product by HPLC pFN300 27.0 32.0 Trace pCZ308 25.0 13.5 500 mg/1 pIClOl 27.0 17.3 600 mg/1

As seen from Table 2, the amount of fusion protein produced by pCZ308 is comparable, although slightly lower than, the amount of Δ7-pST produced by pIClOl. The amount of fusion protein produced was much greater than the amount of pST expressed from pFN300. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Synenki, Richard M. McMullen, James R. Zook, Christopher A.

(ii) TITLE OF INVENTION: PLASMID VECTOR USEFUL FOR THE EXPRESSION OF A FOREIGN DNA SEQUENCE

(iii) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: PATENT DEPARTMENT, PITMAN-MOORE, INC.

(B) STREET: 421 EAST HAWLEY STREET

(C) CITY: MUNDELEIN

(D) STATE: ILLINOIS

(E) COUNTRY: U.S.A.

(F) ZIP: 60060

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/136,148

(B) FILING DATE: 15-OCT-1993

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Ernst, Barbara G.

(B) REGISTRATION NUMBER: 30,377

(C) REFERENCE/DOCKET NUMBER: 0184-1154

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202)783-6040

(B) TELEFAX: (202)783-6031

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CGGGATATCA GCAGCTGGGC C

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(ix) FEATURE

(D) OTHER INFORMATION: COMPLEMENTARY STRAND

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : CAGCTGCTGA TATCCCGGGC C

(2) INFORMATION FOR SEQ ID NO:3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 94 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 : CTAGATGCAT GCTCGAGCGG CCGCCAGTGT GATGGATATC TGCAGAATTC CAGCACACTG GCGGCCGTTA CTAGTGGATC CGAGCTCGGT ACCA

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 94 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(ix) FEATURE

(D) OTHER INFORMATION: COMPLEMENTARY STRAND

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 :

AGCTTGGTAC CGAGCTCGGA TCCACTAGTA ACGGCCGCCA GTGTGCTGGA ATTCTGCAGA

TATCCATCAC ACTGGCGGCC GCTCGAGCAT GCAT

(2) INFORMATION FOR SEQ ID NO:5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 331 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 : AGCTTGGGGG GATCGAAGTT AAAGGTATCA AAGACGTTGT AACTCAGCCG CAGGCTTAAG TTCTCGTCTG GTAGAAAAAC CCCGCTGCTG CGGGGTTTTT TTTGCCTTTC AGTAAATGAA 1 CTGACTTTCG TCAGTTATTC CTTACCCAGC AATGCCTGCA GATCGAAGTT AAAGGTATCA 1 AAGACGTTGT AACTCAGCCG CAGGCTTAAG TTCTCGTCTG GTAGAAAAAC CCCCGCTGCT 2 GCGGGGTTTT TTTTGCCTTT AGTAAATTGA ACTGACTTTC GTCAGTTATT CCTTACCCAG 3 CAATGCCTGC AGATCCGTCG ACCTGCAGCC A 3

(2) INFORMATION FOR SEQ ID NO:6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 331 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(ix) FEATURE

(D) OTHER INFORMATION: COMPLEMENTARY STRAND

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 :

AGCTTGGCTG CAGGTCGACG GATCTGCAGG CATTGCTGGG TAAGGAATAA CTGACGAAAG

TCAGTTCAAT TTACTAAAGG CAAAAAAAAC CCCGCAGCAG CGGGGTTTTT CTACCAGACG 1

AGAACTTAAG CCTGCGGCTG AGTTACAACG TCTTTGATAC CTTTAACTTC GATCTGCAGG 1

CATTGCTGGG TAAGGAATAA CTGACGAAAG TCAGTTCAAT TTACTGAAAG GCAAAAAAAA 2

CCCCGCAGCA GCGGGGTTTT TCTACCAGAC GAGAACTTAA GCCTGCGGCT GAGTTACAAC 3

GTCTTTGATA CCTTTAACTT CGATCCCCCC A 3

Claims

What is claimed is:

1. A universal cloning vector, comprising a DNA sequence comprising from the 5' end to the 3' end: a) a promoter, b) a transcription initiation site, c) a DNA sequence encoding a portion of the N-terminus of Δ7-pST wherein said portion encodes at least about 7 amino acids, and d) a first synthetic oligonucleotide comprising a restriction enzyme recognition site; wherein each of the above are operatively linked to one another, in the order set forth.

2. The vector of claim 1 further comprising a transcription termination site operatively linked to the 3' end of said first synthetic oligonucleotide.

3. The vector of claim 2 wherein said restriction enzyme recognition site is a blunt end site.

4. The vector of claim 1 wherein said plasmid further comprises a second synthetic oligonucleotide, its 5' end operatively linked to the 3' end of said first oligonucleotide.

5. The vector of claim 4 wherein said first oligonucleotide comprises three blunt-end restriction enzyme recognition sites, each of which can be cleaved at a different position within the reading frame of the DNA sequence.

6. The vector of claim 1 wherein said first oligonucleotide comprises the DNA sequence 5' CGG GAT ATC AGC AGC TGG GCC 3' (SEQ. ID. NO:l) .

7. The vector of claim 4 wherein said first oligonucleotide comprises the DNA sequence 5' CGG GAT ATC AGC AGC TGG GCC 3' (SEQ. ID. NO:l) .

8. The vector of claim 7 wherein said second oligonucleotide comprises a sticky-end enzyme recognition restriction site.

9. The vector of claim 8 wherein said second oligonucleotide comprises the DNA sequence

5' CTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCC 3' TACGTACGAGCTCGCCGGCGGTCACACTACCTATAGACGTCTTAAGG

AGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCA 3' (SEQ. ID. NO:3) TCGTGTGACCGCCGGCAATGATCACCTAGGCTCGAGCCATGGTTCGA 5' (SEQ. ID. NO:4)

10. The vector of claim 4 wherein said first oligonucleotide comprises three unique sticky-end restriction recognition enzyme sites, each of which is aligned in a different position within the reading frame of the DNA sequence.

11. The vector of claim 10 wherein said second oligonucleotide comprises a blunt-end restriction enzyme recognition site.

12. The vector of claim 4 further comprising a transcription terminator and a selectable marker.

13. The vector of claim 1 or 6 further comprising a foreign DNA sequence encoding a heterologous polypeptide, said sequence operatively inserted downstream from, and in reading frame with, the DNA sequence encoding a portion of the N-terminus of Δ7- pST.

14. The vector of claim 13 wherein said heterologous polypeptide comprises EGF.

15. The vector of claim 13, wherein said heterologous polypeptide comprises a mammalian somatotropin.

16. The vector of claim 15 wherein said somatotropin is porcine somatotropin.

17. The vector of claim 13 wherein the DNA sequence encoding a portion of the N-terminus of the Δ7-pST gene comprises from about the first 21 sequential base pairs to about the first 60 sequential base pairs.

18. The vector of claim 17 which comprises from about 30 sequential base pairs to about 45 sequential base pairs of the N-terminus of the Δ7-pST gene.

19. The vector of claim 18 which comprises 30 sequential base pairs of the N-terminus of the Δ7-pST gene.

20. The vector of claim 13 wherein said promoter comprises a P_L promoter.

21. Organisms transformed with the vector of claim 1.

22. Organisms transformed with the vector of claim 13.

23. The organism of claim 22 wherein said organism comprises E. coli.

24. The vector of claim 1 which comprises Plasmid pCZ306.

25. A fusion protein comprising the N-terminus of Δ7-pST fused to the amino acid sequence encoding EGF.

26. A fusion protein comprising the N-terminus of Δ7-pST fused to the amino acid sequence encoding somatotropin.

27. The fusion protein of claim 26, wherein said somatotropin is porcine somatotropin.

28. The universal cloning vector of claim 1 comprising a plasmid containing in an operatively linked, sequential manner in the 5' to 3' direction: a) a P_L promoter, b) a transcription initiation site, c) a 30 base pair sequence of DNA encoding the first 10 amino acids of the N-terminus of Δ7-pST, d) a first synthetic oligonucleotide comprising the sequence CGGGATATCA GCAGCTGGGCC, e) a second back-end synthetic oligonucleotide having the DNA sequence

AGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCA 3' (SEQ. ID. NO:3) TCGTGTGACCGCCGGCAATGATCACCTAGGCTCGAGCCATGGTTCGA 5' (SEQ. ID. NO:4) f) a transcription terminator, and g) a selectable marker.

29. E. coli containing the vector of claim 28.

30. The plasmid vector of claim 1 for cloning any gene into any of the three phases of a known reading frame comprising DNA sequences operatively linked together in the following order: a) a P_L promoter, b) a Mu ribosome binding site, c) a transcription initiation site, d) a DNA sequence encoding a portion of the N-terminus of Δ7-pST protein to provide a known reading frame, e) a first synthetic oligonucleotide comprising three blunt end restriction enzyme sites each of which is aligned in a different one of the three phases of said reading frame, f) a second synthetic oligonucleotide comprising at least one sticky-end restriction enzyme site, and g) a transcription termination di er.

31. A oligonucleotide sequence comprising

5' CGG GAT ATC AGC AGC TGGGCC 3'

32. An oligonucleotide sequence comprising:

5' CTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCC 3 ' TACGTACGAGCTCGCCGGCGGTCACACTACCTATAGACGTCTTAAGG

33. A method for expressing a protein in a host cell which comprises:

(a) inserting the DNA sequence encoding the protein of interest into the vector of claim 1, such that the DNA sequence is under the control of the promoter/operator and operatively linked to and downstream from the DNA sequence encoding at least the first 7 amino acids of D7-pST,

(b) transforming a host cell with the vector of step (a) , and (c) culturing the transformed cell under conditions which enable expression of the protein of interest, wherein the protein is produced as a fusion protein, fused to the amino acids of D7-pST.

34. The vector of claim 1 which comprises plasmid pCZ308.