WO1984000380A1

WO1984000380A1 - Vector

Info

Publication number: WO1984000380A1
Application number: PCT/GB1983/000172
Authority: WO
Inventors: John Spencer Emtage; Christopher Clive Ga Hentschel; Paul Edward Stephens
Original assignee: Celltech Ltd
Current assignee: Celltech R&D Ltd
Priority date: 1982-07-13
Filing date: 1983-07-13
Publication date: 1984-02-02
Anticipated expiration: 1986-01-13
Also published as: EP0113372A1

Abstract

An expression vector suitable for the production of fusion proteins. The vector includes a nucleotide sequence comprising in phase form the 5' end; a trp promoter-operator, a trpE ribosome binding site, a structural gene comprising a trpE gene, or a 5' portion thereof and a gene coding for a heterologous polypeptide wherein the nucleotide sequence does not include a trp attenuator. The structural gene may further include a 3' portion of a trpE gene downstream of the gene coding for the heterologous polypeptide. Vectors are described which include a gene coding for human calcitonin.

Description

VECTOR

Fi eld o f the Invention

This invention relates to the field of recombinant DNA biotechnology. In particular it relates to a vector, an organism transformed with the vector, a process for producing a polypeptide, and a polypeptide produced by the process. Background to the Invention

The recent advances in recombinant DNA biotechnology have led to an increased demand for expression vector systems which afford particular advantages for particular desired protein products.

The expression of genes coding for relatively short polypeptides often presents problems as the expression products are in some cases unstable and may prove hard to isolate in significant quantities. It is known to overcome such problems by arranging for the production of a fusion protein comprising a sequence of amino acids homologous with the host organism in which expression is taking place, linked to the desired polypeptide which is heterologous to the host organism. The present invention is concerned with a vector capable of directing the production of such fusion proteins.

The tryptophan operon of E. coli has been the subject of detailed study in recent years. The result of this has been the elucidation of the complete nucleotide sequence of the operon (C. Yanofsky et al Nucleic Acids Research 9 , 6647-6668, 1981). The tryptophan operon (hereinafter referred to as the trp operon or the trp gene) comprises, in sequence; a promoter (including an operator), a transcription start site, an attenuator, and five structural genes known as trpE, trpD , trpC, trpB and trpA (in order of their transcription). Each structural gene has an associated ribosome binding site (Shine and Delgano sequence). The five structural genes code for enzymes which are used in a multi-step biosynthesis of tryptophan from chorismic acid. Expression of the genes coding for the five enzymes is controlled either by repression, resulting from excess tryptophan forming a repressor complex with another molecule, and then binding to a repression site in the promoter region, or by attenuation in the attenuator region of the trp operon. The attenuator region of the trp operon possesses a gene coding for a ribosome binding site which, when transcribed, directs the production of a 14 amino acid 'leader' polypeptide. The mRNA transcript of the gene coding for the leader polypeptide assumes a secondary structure, the conformation of which determines whether the transcription of the structural genes takes place. If tryptophan is abundant, the translation of the leader mRNA is closely linked to transcription of the gene coding for the leader peptide. This causes the mRNA to assume a secondary structure which terminates transcription. If tryptophan is deficient, an alternative conformation of the leader mRNA is formed which permits transcription through the attenuator and through the structural genes (see Miozzari, G.F. and Yanofsky C.J. Bact. 133, 1457-1466, 1978; Lee, F. and Yanofsky, C. Proc. Natl. Acad. Sci. U.S.A. 74, 4365-4369, 1977; Oxender, D. Zurawski, G. and Yanofsky, C. Proc. Natl. Acad. Sci. U.S.A, 76, 5524-5527, 1979).

The control regions and parts of the structural genes have been used to construct vectors for the expression of heterologous polypeptides (B.E. Enger-Valk et al Gene 9 , 69-85, 1980; W. Tacon, N. Carey and J.S. Emtage, Molec. Gen. Genet. 177, 427-438, 1980; R.A. Hallewell and J.S. Emtage, Gene 9 , 27-47, 1980). It has been shown that the control regions and the complete trpE gene can be isolated from the E. coli chromosome on a Hind III fragment having about 5,700 base pairs (A.S. Hopkins, N.E. Murray and W.J. Brammar J. Mol. Biol. 107, 549-569, 1976).

The presence of the attenuator in the trp operon is disadvantageous if the control region of the operon is to be used to express heterologous genetic material. In order to express genetic material an organism requires a plentiful supply of amino acids, including tryptophan. However, the attenuator of the trp operon reduces the transcription of the structural genes in response to high levels of tryptophan. The attenuator only allows abundant expression in response to low levels of tryptophan. Such low levels of tryptophan may result in inefficient translation. This effect is not important in natural E. coli cells but is disadvantageous in a genetically manipulated system, where high expression levels are the object.

Published British patent application GB2073203A describes plasmid vectors including the trp promoter-operator system from which part of the attenuator region has been deleted. The plasmids described comprise the trp promoter-operator, nucleotides coding for the leader peptide ribosome binding site and nucleotides coding for a structural gene. The plasmids contain neither the complete trp attenuator region nor nucleotides coding for the trpE ribosome binding site. Nucleotides have been deleted from a point downstream of the ribosome binding site of the leader peptide to a point in the nucleotide sequence of trpE. The plasmids described rely upon the ribosome binding site of the leader peptide to direct production of a fusion protein comprising, for example, six amino acids of the leader polypeptide, the distal third (C-terminal) of the enzyme coded for by trpE and a heterologous polypeptide. Summary of the Invention

According to the present invention we provide a vector including a nucleotide sequence comprising in phase from the 5' end; a trp promoter-operator, a trpE ribosome binding site, and a structural gene comprising a trpE gene, or a 5' portion thereof and a gene coding for a heterologous polypeptide, wherein the nucleotide sequence does not include a trp attenuator. Preferably the structural gene further includes a 3' portion of a trpE gene downstream of the part of the structural gene coding for the heterologous polypeptide, without an intervening stop codon.

The vector is suitable for the production of a fusion protein comprising the heterologous polypeptide linked to the protein coded for the trpE gene or at least a portion or portions thereof.

The vector is preferably a plasmid vector. The trp promoter-operator, the trpE ribosome binding site, the trpE gene or the 5' portion thereof and where appropriate the 3' portion of the trpE gene may be derived from any bacterium having a trp operon. Preferably, however, they are derived from the E.coli trp operon.

The term 'heterologous polypeptide' as used herein means a polypeptide not normally produced by or found in a bacterial host organism. The term 'in phase' as used herein means in the same translation reading frame.

The '5' portion' of the trpE gene is to be taken as a part of the trpE gene commencing with the 5' end of the gene. The '3' portion' of the trpE gene is here to be taken as part of the trpE gene ending with the 3' end of the gene.

In another aspect of the invention we provide a process for the production of a polypeptide comprising the steps of culturing a host organism transformed with a vector of the invention, to obtain a fusion protein, and cleaving the fusion protein to produce the polypeptide. Preferably the fusion protein is a protein comprising the amino acid sequence coded for by the trpE gene or a 5' portion thereof and the amino acid sequence of the polypeptide. In the alternative the fusion protein may comprise, the amino acid sequence coded for by the trpE gene or a 5' portion thereof, the amino acid sequence of the polypeptide and the amino acid sequence of a 3' portion of the trpE gene.

In another aspect of the invention we provide a host organism transformed with a vector of the present invention. In another aspect of the invention we provide a polypeptide produced by the process of the present invention and we further provide a fusion protein produced as an intermediate in the process of the present invention. Preferably the host organisms transformed with the vector of the invention are grown in the presence of an abundance of tryptophan which has the effect of repressing expression of the heterologous genetic material. When a level of host organisms has been reached which is suitable for industrial production, the level of tryptophan in the culture medium is reduced thereby derepressing expression and allowing production of the polypeptide.

In yet another aspect of the invention we provide a vector including a nucleotide sequence comprising in phase, from the 5' end; a trp promoter-operator, a trpE ribosome binding site, a trpE gene, or at least a 5' portion thereof, and a restriction site suitable for the insertion of a gene coding for a heterologous polypeptide. wherein the nucleotide sequence does not include a trp attenuator.

Preferably the nucleotide sequence included in the vector further includes a 3' portion of a trpE gene, downstream of the gene coding for the heterologous polypeptide. Brief Description of the Drawings

Embodiments of the present invention are described below with reference to the accompanying drawings in which;

Figure 1 - shows a schematic process for isolating a 234 bp Taq I fragment from ptrpE₅₇₀₀ DNA, which contains the trp promoter-operator,

Figure 2 - shows a schematic process for inserting the Taq I fragment of Figure 1 into plasmid pAT153 to form plasmids pCT12 and pCT28,

Figure 3 - shows schematically the preparation of pCT29 from pCT28 and the preparation of pCT29 for the insertion of E.coli trpE gene,

Figure 4 - shows schematically the isolation of the trpE gene from ptrpE₅₇₀₀ DNA and its insertion into pCT29 to form pCT37,

Figure 5 - shows the nucleoticie sequence, of part of the trp operon indicating the deletions made to the sequence to form pCT33, pCT34. and pCT37, Figure 6 - shows a scheme for inserting a gene coding for human calcitonin (derived from plasmid phTB3) into plasmid pCT37,

Figure 7 - shows schematically preparation of deletions of pCTD5, Figure 8 - shows the nucleotide sequence around the Bgl II sites of plasmids designated 10g, 10i and 20a,

Figure 9 - shows a scheme for the insertion of the calcitonin or calcitonin glycine gene into vectors 10g, 10i and 20a. Description of the Invention

The invention is now described with reference to a number of embodiments.

1. Construction of vectors

The control region of the trp gene and the trpE gene were isolated using cosmid vectors and in vitro packaging (B. Hohn and K. Murray Proc. Natl. Acad. Sci. U.S.A. 74, 3259-3262, 1977; Collins, J and Hohn, B. Proc. Natl. Acad. Sci. U.S.A. 75, 4242-4246, 1978). E.coli chromosomal DNA was partially digested with Sau 3A and the resulting fragments ligated to a 10.3 kb cosmid, 3030, that had previously been digested to completion with Bam HI. The cosmid 3030 contains a unique site for Bam HI and confers resistance to ampicillin. After ligation, the mixture was packaged in vitro and used to infect a trpE- strain of E.coli K12. Recombinants were selected on L-agar plates containing ampicillin from which they were replica plated onto M9 salts, glucose, casamino acids minimal agar plates supplemented with ampicillin. Recombinants were thus selected that complemented the trpE- strain by their ability to grow in the absence of tryptophan. Several recombinants were identified and plasmid DNA isolated and characterised by restriction enzyme analysis. One cosmid, designated 3030/trp, producing a 5700 bp fragment on digestion with Hind III was selected for further use. Cosmid 3030/trp was digested with Hind III, the fragments separated by agarose gel electrophoresis and the 5700 bp fragment recovered from a gel slice. Finally, this 5700 bp fragment was cloned into the Hind III site of pAT153 to produce the plasmid ptrp E₅₇₀₀.

A schematic representation of a portion of the tryptophan promoter-containing fragment is given in Fig. 1. From this figure it can be seen that the complete tryptophan promoter-operator complex can be isolated on a Taq I restriction fragment of 234 base pairs (bp) if the DNA is digested under conditions where about 50% of the available sites are cleaved. Thus, 10 μg o f ptrp E _{57 00} was incubated in 10 mM Tris-HCl pH 8.4, 100 mM NaCl, 6 mM MgCl₂ and 6 mM β-mercaptoethanol at 65°C for 20 minutes with 4 units of Taq I. After incubation the reaction mixture was extracted with phenol, precipitated with ethanol, dissolved in water and electrophoresed in a 5% polyactylamide gel. After electrophoresis the gel was stained with ethidium bromide and the 234 bp DNA band excised from the gel and the DNA recovered ( A.M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci U.S.A. 74, 560-564, 1977).

This DNA fragment was then inserted into the Cla I site of the plasmid pAT153 (Twigg and Sherratt, Nature 283, 216, 1980). 10 μg of pAT153 was digested to completion with Cla I and the 5'- phosphate groups removed by incubation at 37°C for 1 hour with 0.5 units of calf intestine alkaline phosphatase in 10 mM Tris-HCl, pH 8. This procedure prevents the plasmid recircularising in the absence of an inserted DNA fragment. 100 ng of the above treated pAT153 was ligated at 15°C for 4 hours to 5 ng of the 234 bp DNA in a reaction containing 50 mM Tris-HCl pH 7.6, 10 mM MgCl₂, 20 mM dithiothreitol, 1 mM ATP and 20 units of T4 DNA ligase. The ligated DNA was then used to transform competent E.coli K-12 strain HB101 (Boyer H.W. and Roulland Dussoix, D. J Mol. Biol. 41, 459-472, 1969) by standard techniques (Hershfield, V. et al Proc. Natl. Acad. Sci. U.S.A. 71, 3455-3459, 1974) and the bacteria plated on L-agar plates containing 100 μg/ ml ampicillin. Several ampicillin resistant colonies were selected, plasmid DNA prepared and the presence of the 234 bp fragment confirmed by restriction analysis. The resulting plasmid, designated pCT12, has the structure shown in Fig. 2. Insertion of the Taq fragment into the Cla I site reforms a Cla I site at the end downstream from the trp promoter. pCT12 was further modified to produce pCT28 and pCT29 as follows. 2 μg of pCT12 was digested to completion with Hind III and then incubated at 20°C for 30 minutes with 60 units of SI nuclease in a buffer containing 25 mM sodium acetate, pH 4.5, 0.3 M NaCl and 1 mM zinc acetate to remove the protruding Hind III ends. The reaction was terminated by raising the pH to 7.6 and extracting the mixture with phenol-chloroform (1:1). The DNA was con centrated by ethanol precipitation and a sample incubated with T4 DNA ligase as described above and then used to transform E.coli K12 strain HB101. Several ampicillin resistant clones were selected, plasmid DNA prepared and the absence of the Hind III site confirmed by restriction analysis. The resulting plasmid, designated pCT28, has the structure shown in Fig. 2. pCT28 was further modified. 2 μg of EcoRI-digested pCT28 was incubated with 4 units of E.coli DNA polymerase for 15 minutes at 10°C in a reaction containing 50 mM Tris-HCl pH 7.6, 10 mM MgCl₂, 10 mM β-mercaptoethanol,

0.2 mM dATP and 0.2 mM TTP. After incubation the mixture was extracted with phenol, with chloroform and then ethanol precipitated. This treatment causes the 4 nucleotides complementary to the 5' protruding ends of the EcoRI site to be filled in:-

5' A A T T C - 5' A A T T C - 3' G - 3' T T A A G -

0.6 μ g o f th e abov e treated pCT28 was treated wi th 50 units of T4 DNA ligase in the presence of 30 picomoles of the 5' -phosphorylated synthetic oligonuclotide pCCAAGCTTGG and in 10 μl T4 DNA ligase buffer at 15°C for 16 hours. The mixture was then heated at 70°C for 10 minutes to stop the reaction and the linkers cleaved by digestion with Hind III. The linear DNA, now with Hind III ends, was separated from the linkers by electro phoresis on an agarose gel from which it was subsequently recovered by electroelution and concentrated by ethanol precipitation. Ligation, followed by transformation of E.coli K12 strain HB101, isolation of plasmid DNA and identification of plasmids with Hind III and EcoRI sites, produced the plasmid designed pCT29. pCT29, Fig. 3, contains the E.Coli tryptophan promoter-operator region and, as well, unique restriction sites for the enzymes Cla I, Hind III and EcoRI downstream of the promoter region. Two of these sites, the Cla I and Hind III sites, were used to insert the trpE gene.

5 μg of pCT29 was cleaved with Cla I and the 5'-protruding ends filled in as described above using E.coli DNA polymerase in the presence of dCTP and dGTP. The polymerase reaction mixture was extracted with phenol, then with chloroform and the DNA finally precipitated with ethanol. The filled-in DNA was then digested with Hind III and the resulting fragments separated by agarose gel electrophoresis. The largest fragment was isolated from the gel by first staining with ethidium bromide, locating the DNA with ultravi ol et light and cutting from the gel the portion of interest. The DNA was recovered from the gel fragment by electroelution and concentrated by efchanol precipitation. The trpE gene is present on the plasmid ptrp E₅₇₀₀ , a portion of which is shown in Fig. 4. As described above however , ptrp E₅₇₀₀ also contains the trp attenuator. To remove the attenuator region 5 μg of ptrp E₅₇₀₀ was digested with Hpa I and then treated with nuclease BAL 31. This nuclease isahighly specific nuclease that can be used to shorten DNA fragments from the ends. Further, its action produces blunt-ended molecules. 5 μ g of Hpa I digested ptrp E₅₇₀₀ was treated with 1.5 units of BAL 31 nuclease in 0.6 M NaCl, 12 mM CaCl₂, 12 mM MgCl₂, 20 mM Tris-HCl pH 8, 1 mM EDTA at 30°C for 1.5 minutes. The mixture was then phenol extracted, chloroform extracted and ethanol precipitated. This treatment removes 150-250 bp from each end of the DNA fragments and so will remove the attenuator region from most molecules as it is 150 bp from the Hpa I site. The above treated DNA fragments were digested to completion with Hind III and then separated by agarose gel electrophoresis. DNA containing the trpE gene was isolated from the gel by first staining the gel with ethidium bromide, locating the DNA with ultraviolet light and cutting from the gel DNA in the size range 1800- 1850 bp. This DNA was recovered from the gel fragment by electroelution and concentrated by ethanol precipitation.

The trp E fragments isolated above can be inserted into pCT 29 that had been modified as described above. Fig. 4 illustrates the ligation reaction. 0.2 μg of modified pCT29 and 80 μ g of the trpE fragment were incubated at 20°C for 16 hours with 100 units T4 DNA ligase in 50 mM Tris pH 7.6, 10 mM MgCl₂, 1 mM ATP and 20 mM-dithiothreitol. The mixture was then used to transform E.coli K12 strain HB101 as described above and trans formants selected on L-agar plates containing ampicillin. Several ampicillin resistant colonies were selected, plasmid DNA isolated and examined by restriction analysis. This analysis confirmed the presence of the trpE gene and the loss of the attenuator region. Three plasmids were isolated and were designated pCT33, pCT34 and pCT37.

Nucleotide sequence analysis was performed using the dideoxy chain termination method (Sanger et al . , 1977 PNAS, 74, 5463-5467). The analysis showed that nucleotides 24 to 131, inclusive, had been lost in pCT33 and pCT37 and that nucleotides 24 to 151, inclusive, had been lost in pCT 34, (assuming that the start point of mRNA transcription is nucleotide 1). This further confirmed that all of the attenuator region of trpE had been deleted in these plasmids (Fig. 5).

Plasmid pCT37, was selected for further work involving the human clacitoπin gene. (This work is described in Published International Patent Applications WO 83/00327 and WO 83/00346).

2. Calcitonin and Calcitonin-Gly Fusions with trp E

The scheme outlined in Fig. 6 shows the steps carried out to combine the human calcitonin sequence derived plasmid phT-B3 (see above mentioned published International Patent Applications) with the expression vector pCT37 described above to produce fusion proteins. The initial step involves the modification of the calcitonin sequence so that the ultimate amino acid in the fusion protein is either the proline corresponding to the authentic terminal amino acid in calcitonin or so that a further glycine is translated. The purpose of this construction relates to the final processing envisaged in calcitonin production and is described fully below. 20 μg of phT-B3 were incubated in 6 mM Tris-HCl pH 7.5, 6 mM MgCl₂, 6 mM β-mercaptoethanol and 20 mM KCl with 10 units of Bst NI for 60 minutes at 60°C. After incubation the reaction was made 0.3 M in NaAcetate pH 6:0, extracted with phenol, then chloroform and concentrated by ethanol precipitation. The precipitated DNA was washed with 70% ethanol, dried under vacuum and redissolved in 20 μl of water (Step i Fig. 6). Bst NI cuts the calcitonin sequence only in the proline at position 32 and in such a way that the T residue in the anti-coding strand is removed. This T residue is added back in the next step (ii). 10 μg of the Bst NI cleaved phT-B3 were incubated in 60 mM Tris-HCl pH 7.6, 10 mM MgCl₂, 10 mM p-mercaptoethanol, 0.2 mM dCTP, 0.2 mM dTTP and 6 units of Klenow enzyme (DNA-polymerase large fragment; supplied by the Boehringer Corporation (London) Ltd.). After the incubation the DNA was phenol extracted, chloroform extracted, ethanol precipitated, washed in 70% ethanol and redissolved in 10 μl of water. The resulting DNA is now flush-ended and can be ligated to

other flush-ended DNA molecules. The DNA was ligated (covalently joined) with two separate synthetic oligc nucleotides designed A or B in Fig. 6:-

A = T A G G A T C C T A A T C C T A G G A T

B = G G T T G A T C A A C C C C A A C T A G T T G G

Thus 2.5 μg of the phT-B3 derived DNA fragments were incubated in separate reactions with 400 ng of oligo nucleotides A or B in 60 mM Tris-HCl pH 7.5, 8 mM MgCl₂,

10 mM β-mercaptoethanol, 1 mM ATP and 1.5 μl T4 DNA ligase (New England Biolabs Lot 17) for 24 hours at 16°C. Ligase activity was destroyed by raising the temperature to 70ºC for 5 minutes and the DNA ethanol precipitated, washed in 70% ethanol, dried under vacuum and redissolved in 10 μl of water. 1 μl of both DNA samples (i.e. ligated with oligonucleotiede A or B) was analysed for efficient lig ation, as evidenced by its increase in size through con catemerisation and visualised by staining an agarose gel with ethidium bromide following an electrophoretic size separation. Note that the two ligation reaction samples represent the start of two separate and parallel construction routes (i.e. A or B) as shown in Fig. 6. The description of the experiments below apply for both series of constructions. The remaining plasmid DNA (9 μl ) was separated from unreacted oligonucleotides by Sephadex G-50 chromatography and concentrated by ethanol precipitation. The DNA was redissolved in 50 ul 6 mM Tris-HCl, 50 mM NaCl, 6 mM MgCl₂, 6 mM B-mercaptoethanol and excess Sau 3A enzyme added to ensure complete cutting at all Sau 3 sites after 1 hours at 37°C. The linker oligonucleotides contain the Sau 3A recognition sequence 5 ' GATC 3' and the Bgl II site 5' AGATCT 3' is also a Sau 3A site so that after the Sau 3A digestion, the calcitonin sequence resides in DNA fragments about 110 base pairs long (the exact length differing by one nucleotide depending on whether oligonucleotide A or B was used). These fragments were isolated following polyacrylamide gel electrophoresis (PAGE) as described above (Step iv Fig.6) and following ethanol precipitation, were dissolved in water (10 μl ) . The vector expression plasmid pCT37 (described in detail above) was prepared for ligation into the Bgl II site as follows. 10 μg of pCT37 was incubated in 6 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl₂, 6 mM β-mercaptoethanol and 10 units Bgl II for 60 minutes at 37°C. The linearised DNA was diluted to 0.5 ml and made 0.3 M in sodium acetate pH 6.0. 5' Phosphate groups were removed by the addition of 3 units of calf intestinal alkaline phosphatase (CIAP) and incubation at 60°C for thirty minutes, after which a further 3 units of CIAP was added and a further 30 minute incubation carried out.

Phosphatase activity was destroyed by one phenol, two phenol-chloroform and three chloroform extractions, after which the DNA was ethanol precipitated and redissolved in water (10 μl). In Step vi the calcitonin containing fragments were ligated into the Bgl II site of p CT37 as follows. 0.1 μg of the Bgl II linearised and CIAP treated pCT37 was incubated with approximately 2 ng of the calcitonin containing fragments in 60 mM Tris-HCl pH 7.5, 8 mM Mgcl₂, 10 mM β-mercaptoethanol, 1 mM ATP and 0.2 μl T4 DNA ligase (New England Biolabs Lot 17) for 16 hours at 16°C. The DNA was then used to transform frozen competent E.coli HB101 using standard methods (cf Methods in Enzymology Vol 68, pp 326-331) and transformants resistant to ampicillin selected on L-agar plates containing 100 μg/ml ampicillin. Clones containing plasmids with the calcitonin sequence in the Bgl II site and in the correct orientation were identified and the constructions confirmed by DNA sequencing using the method of Maxam A and Gilbert, W Proc. Natl. Acad. Sci. 74 560 (1977). At this stage the calcitonin DNA translation reading frame is out of phase with the trpE gene reading frame and if this reading frame is being used by a ribosome, a stop codon (TAA) will be encountered after amino acid 322 of the trpE gene. Thus in contrast to the parent expression vector pCT37 which directs the overproduction of trpE gene product, these constructions would be expected to direct the overproduction of a novel truncated protein 323 amino acids long (i.e. about 352. Daltons M Wt).

That such a novel protein is indeed produced by E.coli harbouring these plasmids, and only under appropriate inducing conditions, has been established experimentally. Thus the final steps in the construction of plasmids directing the overproduction of the desired trpE-calcitonin (or calcitonin-gly) fusion proteins requires the calcitonin sequence to be brought into the same translation reading frame as the trpE gene This can be achieved by adding 3n + 2 nucleotides at the fusion junction between the trpE sequence and the calcitonin sequence (i.e. at the recreated unique Bgl II site). The synthetic octomer

5' G A T C C C G G

G G C C C T A G is suitable since it is of the correct length (8 = 3 x 2 + 2), has an appropriate 5' overhang for ligation into the Bgl II site and in addition creates a new unique restriction site for the enzyme Sau (5' CCCGGG 3') at the fusion junction. Thus the plasmids (10 μg) were incubated in 6 mM Tris-HCl, 50 mM NaCl, 6 mM β-mercaptoethanol, 6 mM MgCl₂ with 10 units Bgl II and the phosphate groups removed from the linear molecules using CIAP as described above. Following phenol extraction and ethanol precipitation 0.1 μg of the linear DNA was incubated with a 10-fold molar excess of the synthetic oligonucleotide G A T C C C G G

G G C C T A G in 60 mM Tris-Hcl pH 7.5, 8 mM MgCl₂, 10 mM β-mercaptoethanol, 1 mM ATP and 0.2 μl T4 DNA ligase, for 16 hours at 16°C (Step ix Fig. 6). The ligation products were used to transform E.coli HB101 cello and amplicillin resistant transformed cloned isolated on L-agar plates containing 100 μg/ml ampicillin.

The majority of plasmids in these clones were found to contian more than one copy of the synthetic oligonucleotide at the fusion junotion. 1 μg of plasmids with 2 inserted linkers was linearised by incubation in 6 mM Tris-HCl, 6 mM MgCl₂, 6 mM β-mercaptoethanol and 20 mM KCl with 10 units of Sma I (supplied by the Boehringer Corporation (London) Ltd.), and following purification of the linear molecules from unlinearised plasmid, the DNA was religated with T4 DNA ligase and following a further round of transformation into HB101 plasmids with the structure shown at the bottom of Fig. 6, were isolated. As illustrated the DNA junction is now such that the calcitonin sequence is in phase with the trpE sequence and such plasmids therefore direct the production of a novel fusion protein of the expected size (about 38K Daltons M Wt; see Fig. 10). Verification that E.coli expressing this protein is expressing a human calcitonin sequence has been obtained by radio-immunoassay. C. CALCITONIN FROM A TRP E-CALCITONIN FUSION PROTEIN

The ultimate aim of introducing the human calcitonin sequence into the trp E gene of E.coli in such a way that substantial quantities of a trp E- calcitonin fusion protein are produced is to liberate the calcitonin peptide in a commercially viable way. This may be done by several possible routes. As a first step the calcitonin peptide must be cleaved from the fusion protein. This can be achieved by the use of the enzyme trypsin which will cleave exactly at the fusion junction since the initial cysteine residue is preceded by an arginine. Calcitonin contains no arginines but one lysine residue at position 18 which would also be liable to trypsin cleaveage. This lysine can however be protected by citraconic anhydride (Shine S, Fettes I, Nancy C.Y. Lan, Roberts, J.L. and Baxter, J.D. Nature 285, 456-461). The peptide liberated by this procedure differs from authentic calcitonin only in that in authentic calcitonin the C-terminal amino acid is a prolinamide rather than a proline (or proline-glycine) amino acid. Conversion of the liberated peptide into authentic calcitonin is possible through the use ofthe C-terminal modification activity of yeast carboxypeptidase Y (Breddam, K, Widmer, F and Johanson, J.T. Carlsberg Res. Commun. 45, 237-247 and 361-367 1980).

D. CONSTRUCTION OF VECTORS HAVING TRUNCATED TRP E GENES

PCT37 makes use of a naturally occurring Bglll site in the trp E gene to form a fusion between the N terminus of the trp E gene product and calcitonin. This construction was used as the basis of constructing a further family of trp E fusion vectors in which the 3' end of the trp E gene has been deleted. The trp E-calcitonin fusion plasmid (pCTD5) was linearised with the restriction enzyme Smal (Fig.7). 20μg of pCTD5 was incubated with 20 units of Smal for 2 hours at 37°C in 20mMKCl, 6mM Tris-HCl (pH8.0), 6mM MgCl₂, 6mM β-mercaptoethanol, 100 μg/ml bovine serum albumin. The reaction was stopped by phenol extraction followed by ethanol precipitation. The linear DNA (200ug/μl) was then digested with Bal31. 10μg of DNA was digested with 1.25 units of Bal31 for varying time periods at 30°C in 600mM NaCl, 12mM Ca Cl₂ 12mM MgCl₂, 20mM Tris HCl (pH8.0), 1.0mM EDTA in a total volume of 50μl. 10μl aliquots were removed after 10 minute intervals and the reaction stopped by phenol extraction. The 5' terminal phosphatase groups were removed by the addition of 3 units of calf intestinal alkaline phosphatase (CIAP) and incubation at 37°C for 30 mins, phosphatase activity was destroyed by two phenol and two ether extractions. 100μg of the Bal31 digested DNA was incubated with 25μg of the kinased synthetic oligonucleotide linker R53 (CAAAAGATCTTTTG) in 60mM Tris HCl (pH7.5), 8mM MgCl₂, 10mMβ-mercaptoethanol, ImM ATP and 1.5μl of T4 DNA ligase. (New England Biolabs Lot 20) for 16 hours at 16°C. The synthetic linker introduces a unique Bgl II site into the plasmid. Ampicillin resistant colonies were selected on L.agar plates containing 100μg/ml ampicillin following transformation into frozen competent E.coli HB101 (Methods in Enzymology Vol.68 pp 326-331). The presence of Bglll restriction enzyme site in these plasmids was confirmed by restriction analysis.

The ability of these plasmids to express a novel protein band of truncated trp E was investigated in the following way. E.coli cells bearing a plasmid with a Bglll site were grown at 37°C in either L-broth in which the trp promoter is not induced or in minimal medium (based on M9 salts) in which the promoter is induced (R.A. Hallewell and J.S. Emtage, Gene 9 27-47, 1980). The cells were harvested by centrifugation and dissolved in SDS sample buffer (Laemmli U.K., Nature 227, 580,1970). Protein equivalent to 50μl of original culture was electrophoresed on a 12.5% (W/V) polyacryla mide gel and the separated proteins visualized by staining with Coomassie blue. In this way three plasmids were identified which gave truncated trpE proteins on induction.

The three plasmids 10g, 10i and 20a produced an induci ble trpE protein band on Coomassie blue stained poly acrylamide gels of Mr 30K, 40K and 20K respectively. Restriction enzyme analysis indicated that the remaining N. Terminal portion of the trpE gene in these plasmids is 470 base pairs, 750 base pairs, and 380 base pairs respectively. In all cases the 5' portion of the gene is too small to encode the protein band seen on the Coomassie blue stained gel, and indicates that the trpE translation phase continues across the Bglll site. DNA sequence analysis of these constructions confirms this and indicates in the case of 10g and 10i that the trpE translation phase is maintained across the Bglll site and read through continues to the natural translation stop signal of the trpE gene.

The plasmid 20a, possesses two Bglll sites which generate a fragment of 690 base pairs. This fragment originated from the 5' region of the trpE gene (coordinates 140 to 160 inclusive; numbered according to Yanovsky et al., 1981, N.A.R. 9 6647-6668). The orientation of this fragment in the plasmid was not determined. The nucleotide squence analysis indicates that the phasing around the Bglll sites in these plasmids is different in each case. (Fig.8). The phasing around the Bglll site in 10i is the same as that in the first Bglll site of the authentic trpE gene. The three novel plasmids provide a series of vectors which will allow the cloning of any gene in phase with the truncated N. terminus of trpE in order to produce a fusion protein.

The formation of the trpE calcitonin fusions in the three vectors follows three paths depending on the phasing around the Bglll sites and these are outlined in (Fig.9).

The DNA encoding the human calcitonin gene is provided by cleavage of a precursor of the original trpE calcitonin fusion construction (in which the trpE and calcitonin are out of phase) with Bglll and PstI. 10μg of plasmids D13, (trpE-calcitonin) and E2, (trpE-calcitonin-gly) were individually incubated with 10 units of Bglll and Pstll in 60mM NaCl, 10mM Tris-HCl (pH7.4), 10mM Mgcl₂, 10mM β-mercaptoethanol, 100μg/ml bovine serum albumin at 37°C for 3 hours. Following electrophoresis in low melting point agarose a 1.6Kb fragment containing the calcitonin gene sequence, was purified tythreephenol extractions after the appropriate gel slice had been melted at 70°C for 10 minutes. The fragment was then concentrated by ethanol precipitation. The three vectors were digested with Bglll and Pst as described above. The 5' phosphate groups from the linear fragments were removed by CIAP treatment . 250 μg of cut vector were incubated with a 3M excess of the Bglll-PstI fragment from D or E2 in the presence of 60mM Tris HCl (pH7.5), 8mM MgCl₂, 10mMβ-mercaptoethanol, 1mM ATP and 1.5μl of T4 DNA ligase (New England biolabs, LOT 19) for 16 hours at 16°C. The DNA was then used to transform competent HB101 and transformants were selected for on L.agar plates containing 100μg/ml ampicilin. Clones containing the calcitonin sequence were identified by restriction enzyme analysis. In only one case (i.e. the 20a construction) was the phasing correct to produce a trpE- calcitonin fusion.

In order to get the phasing correct, in the other two cases, the following steps were performed. The 10g construct was linearised by cleavage with Bglll and the 5' overhangs were then filled in. 800μg of plasmid was incubated with .2mM dGTP, .2mM d ATP, .2mM dTTP and 0.2,M dCTP, 10mM Tris-HCl (pH8.5) 10mM MgCl₂, and 2.5 units of E.coli DNA polymerase (large fragment Ace Klenow) at 20°C for 30 min. The reaction was stopped by heating to 70°C for 10 min. The blunt ended linear molecule was then religated using T4 DNA ligase. Ampicillin resistant ransformants were screened for plasmids possessing a unique ClaI site indicating successful filling in of the GAT 5' overhang. The calcitonin should now be in phase with the trpE gene.

After linearising the 10i construct with Bglll the plasmid was incubated with a 100 fold molar excess of synthetic olignucleotide R70 (GATCCCGG) in 60mM Tris HCl, 8mM MgCl₂, 10mMβ-mercaptoethanol, 1mM ATP and 2μl of T4 DNA ligase for 16 hours at 16°C. The ligation products were used to transform E.coli HB101 cells and ampicillin resistant clones were isolated on L.agar plates containing 100μg/ml ampicillin. The plasmids in these clones were screened for the presence of a unique Smal site, indicating the presence of the linker and the corresponding correct phasing between trpE and calcitonin. The production of a trpE calcitonin fusion was confirmed by Western blotting using an antibody raised to authentic human calcitonin (Towbin et al, 1979 PNAS 76 4350). Each construction produced a band of the predicted molecular weight which was immunogenic.

Claims

CLAIMS :

1. A vector including a nucleotide sequence comprising, in phase, from the 5' end; a trp promoter-operator, a trpE ribosome binding site, and a structural gene comprising a trpE gene, or a 5' portion thereof, and a gene coding for a heterologous polypeptide, wherein the nucleotide sequence does not include a trp attenuator.

2. A vector including a nucleotide sequence comprising in phase from the 5' end; a trp promoter-operator, a trpE ribosome binding site, and a structural gene comprising a trpE gene, or a 5' portion thereof, a gene coding for a heterologous polypeptide, and a 3' portion of a trpE gene, without an intervening stop codon, wherein the nucleotide sequence does not include a trp attenuator.

3. A process for the production of a polypeptide comprising the steps of; culturing a host organism transformed with a vector according to claim 1 or 2, to obtain a fusion protein, and cleaving the fusion protein to obtain the polypeptide.

4. A polypeptide produced by a process according to claim 3.

5. A fusion protein produced as an intermediate in a process according to claim 3.

6. A host organism transformed with a vector according to claim 1 or 2.

7. A vector including a nucleotide sequence comprising in phase from the 5' end; a trp promoter-operator, a trpE ribosome binding site, a trpE gene, or at least a 5' portion thereof, and a restriction site suitable for the insertion of a gene coding for a heterologous polypeptide, wherein the nucleotide sequence does not include a trp attenuator.

8. A vector including a nucleotide sequence comprising in phase from the 5' end; a trp promoter-operator, a trpE ribosome binding site, a trpE gene or at least a 5' portion thereof, a restriction site suitable for the insertion of a gene coding for a heterologous polypeptide, and a 3' portion of a trpE gene, wherein the nucleotide sequence does not include a trp attenuator.