GB2033905A - Bacterial preparation of proteins - Google Patents

Abstract

Animal or plant high molecular weight proteins can be prepared by a host such as E. coli by using DNA recombinant technology to fuse a gene coding for the protein near transcriptional and translational regions in a vehicle such as a plasmid which is inserted in the host. New plasmids and E. coli strains are described.

Description

SPECIFICATION Bacterial preparation of proteins Many of the potential benefits envisaged as a result of the application of recombinant DNA technologyto medical problems require the insertion, into bacteria, of genes able to direct the biosynthesis of required proteins. In most cases, a protein of interest will normally be synthesised in animal cells and will not be found naturally inE. coli or other prokarvotes. Although it has been possible to clone a number of different animal genes containing the information necessary to code for proteins, reports of the expression of these proteins in bacteria and other unicellular organisms have been limited to the human polypeptide hormone somatostatin (see Itakura etal, Science 198 (1977) 1056-1063) and rat proinsulin.

In the given reference, it is reported that the somatostatin gene has been chemically synthesised and the colding sequence for its 14 amino acids has been fused to the p-galactosidase structural gene on a plasmid. Yields of somatostatin varied from 0.001 to 0.03% of the total cellular protein. The rat proinsulin gene has been inserted into a plasmid-borne penicillinase gene and the bacteria produce approximately 100 molecules of proinsulin per cell.

According to the present invention, a process for the expression of a gene coding for a high molecular weight protein in a suitable vehicle comprises taking the gene and fusing it neartranscriptional and translational initiation regions present in the vehicle while maintaining the translational reading frame, and inserting the vehicle into a suitable host.

Utilising recombinant DNA methodology described below, we have fused the chicken ovalbumin structural gene to E. coli transcriptional and translational control regions. When a plasmid containing the hydrid gene is introduced into E. coli, a protein identified as ovalbumin by immunoreactivity and SDS-polyacrylamide gel electrophoresis is synthesised. The chicken ovalbumin made in bacteria is full-length (43,000 MW) and constitutes 1.5% of the cellular protein (approximately 20,000 molecules per cell). This is the first high molecular weight animal protein expressed in E. coli. It is an advantageous aspect of this process that the egg ovalbumin is secreted through the bacterial cell membrane.

Examples of other high molecular weight proteins which are within the scope of the present invention are human serum albumin, human interferons, human antibodies, blood clotting factors, enzymes, viral antigens and proteins from plants (excluding fungi). On a molecular weight number basis, a high molecular weight protein is herein defined as one having a molecular weight in excess of about 10,000 daltons.

Examples of other vehicles which can be used in the invention are pBR 322 and pBR 313 which code for ampicillin and tetracycline resistance, pSC 101 which codesfortetracycline resistance, pCR 11 which codes for kanamycin resistance, A bacteriophage vectors such as charon phages, and yeast 2 cm plasmid DNA.

Examples of other hosts for the vehicle are any E.

coli K-12 derivative (Bacteriological Reviews, Dec.

1972, pages 525-557) (these have been approved by the NIH Guidelines in the U.S.A.) and yeasts, other fungi, and other bacteria. It is recognised that these latter hosts would also have to be approved. All the restriction endonucleases used herein can be obtained from New England Biolabs, Beverly, Massachusetts, U.S.A.

The process of the invention can be used for plant and animal proteins, and genes from plant and animal viruses. The genes may be isolated from vertebrates, and particularly warm-blooded vertebrates such as birds. The invention is especially suitable for genes from chickens.

The accompanying drawing depicts the process steps for making the lac-ovalbumin-fused plasmid pUC 1001. Although the abbreviations used are conventional and well known to those skilled in the art, they are redefined here to facilitate a clear understanding of the invention.

Restriction endonucleases: Taq I, EcoRI (in the Examples). Hind III, Taq I and EcoRI - restriction endonuclease cleavage sites (in the drawing).

Tc'-tetracycline resistance gene.

dA:dT- deoxyadenosine:deoxythymidine.

p-phosphate.

C - cytosine.

G - guanosine.

A- adenosine.

T- thymidine.

T4 DNA ligase - enzyme coded for by bacteriophage T4.

T4 DNA polymerase - enzyme coded for by bacteriophage T4.

lac - lactose operon control region.

OH - hydroxyl.

pUC-a plasmid owned by The Upjohn Company.

pOP - plasmid operator promotor.

pOV- plasmid ovalbumin.

ovalbumin - chicken ovalbumin structural gene.

The plasmids described herein have been deposited in E. coli hosts in the permanent collection of the Northern Regional Research Laboratory, U.S.

Department of Agriculture, Peoria, Illinois, U.S.A.

Their accession numbers in this repository are as follows: HB101 -NRRLB-11371 HB101 (pOV 230) -NRRLB-11354 HB101 (pOP 203 -NRRLB-11355 HB101 (pUC 1001) - NRRL B-11356 Construction of the lac-ovalbumin fused plasmid, pUC 1001 proceeds as shown in the drawing. The pOP 203 plasmid is cut with EcoRI and the resulting linear molecule is treated, advantageously, with alkaline phosphatase to remove the 5' phosphate groups on the ends of the molecule.

The pOV 230 plasmid contains nearly all ofthe ovalbumin mRNA sequence, including all of the information required to code for the amino acid sequence of chicken ovalbumin[McReynolds etal (1977) Gene2: 217-231]. However, pOV 230 does not express detectable levels of chicken ovalbumin. This is so because it needs a promotor plus a protein synth esis initiation site directed into the ovalbumin struc tural gene. The sequence of the ovalbumin gene insert in pOV 230 [McReynolds etal, Nature (1978) 273: 723-728] has revealed a unique Taq I restriction endonuclease site 25 base pairs to the 5' side of the ovalbumin protein synthesis initiation codon.As shown in the drawing, an additional Taq I site is located approximately 250 base pairs outside of the ovalbumin insert such that Taq I digestion yields a DNA fragment of about 2200 base pairs containing the entire ovalbumin structural gene. At least 11 additional Taq I sites are present in the pOV 230 plasm id, so that digestion yields 12 fragments.

Since the sequence of the lac control region inserted into pOP 203 [Maizels, N. M. (1973) Proc.

Nat. Acad. Sci. 70:3585-3589 and Dickson etal, (1975) Science 187: 27-35] and of the ovalbumin gene insert in pOV 230 is known, the goal is to fuse the lay and ovalbumin DNA's so as to maintain the translational reading frame. This is done by filling in the stagger-ended Taq I fragments with DNA polymerase and then ligating synthetic EcoRI octamer linkers to the blunt ends as shown in the drawing. A large excess of linker molecules is used in the reaction in order to prevent ligation of the filled-in Taq I fragments to each other.

The linked fragments are digested with EcoRI to generate 5' staggered ends. Prior to EcoRI digestion, the DNA fragments are subjected to Hha I restriction endonuclease digestion. This is done, advantageously, to facilitate the subsequent gel purification of the ovalbumin DNA fragment since it does not contain any Hha I sites and many of the other Taq I fragments do.

After elution from the gel, the purified pOV 230 DNA fragment is ligated to the alkaline phosphatase treated pOP 203 plasmid vector as shown in the drawing. This ligated DNA is then used to transform E. coli HB101 NRRL B-11371 and tetracycline resistant colonies are selected.

The tetracycline resistant transformants are assayed for ovalbumin production by an in situ immunoassay. The transformant colonies are lysed with lysozyme and sarkosyl after growing up on agar containing ovalbumin antiserum, IPTG (isopropyl -ss - D - thiogalactoside - available from Sigma Chem.

Co.) and tetracycline. Easily discernible precipitin rings are formed around the colonies containing those plasmids directing the synthesis of ovalbumin.

Cell extracts are made from the ovalbuminproducing transformants. After it is demonstrated that these extracts have immunoreactive ovalbumin, antibody precipitations are done and the precipitates run on an SDS-polyacrylamide gel. The gel analysis shows that the immunoreactive ovalbumin is fulllength (43,000 MW) and no lower molecular weight fragments are observed.

The following Examples are illustrative of the pro cess 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 otherwise noted.

Example 1-DNA Preparation pOP 203 Plasmid DNA from NRRL B-1 1355, is iso lated by the salt precipitation technique described by Guerryetal (1973) J. Bact. 116:1064-1066. L broth [Lennox, E. S. (1955) Virology 1:190-206] containing 10,ag/ml tetracycline is inoculated with an overnight broth culture of NRRL B-11355. Cultures are shaken vigorously at370C. until the optical density at 600 nm reaches 0.8; plasmid copy number is then amplified for 18 hours with chloramphenicol (250 szg/ml). Cells are washed once in 50 mM Tris HCI, pH 8.0,20 mM EDTA, and resuspended in 33 ml of 25% sucrose in TE (10 mM Tris HCI, pH 8.0,0.1 mM EDTA) per liter of culture. Following the addition of 1 mg/ml lysozyme, the suspension is incubated on ice for five minutes, followed by addition of 1/3 volume 0.25 M EDTA, pH 8.0 and another 5 minute incubation on ice. Cells are lysed by addition of 10% sodium dodecyl sulfate (SDS) in 37 mM Tris HCI, pH 8.0,67 mM EDTA, to a final concentration of 1.3% followed by incubation at 370C. for 30 minutes.

Chromosomal DNA is salted out by bringing the NaCI concentration to 1 M; followed by cooling at 4"C. overnight. SDS and chromosomal DNA are removed by centrifuging at 17,000 x g for 30 minutes at 4"C. The resulting supernatant is ethanol precipitated, pelleted, and redissolved in TE. This material is phenol extracted twice, ether extracted, ethanol precipitated, pelleted and resuspended in TE.

Further purification is achieved by cesium chloride-ethidium bromide density gradient centrifugation as described infra.

pOV 230 Plasmid DNA is isolated by the "snap back" procedure described in detail by Currier and Nestor, (1976) Anal. Biochem. 76: 431-441.Lennox broth (1% tryptone, 0.5% yeast extract, 0.5% NaCI, 0.1% glucose) plus 15,g tetracycline per ml are inoculated with an 18 hour broth culture ofE coli HB101 (pOV 230), NRRL B-11354. When the optical density at 600 nm reaches 0.8, plasmid copy number is amplified for 20 hours by addition of chloramphenicol (final concentration of 250 pglml).

Cells are washed twice with 50 mM Tris HCI, pH 8.0,20 mM EDTA, and resuspended in the same buffer at a ratio of 11.25:1 (ml:gram wet weight). Bacteria are lysed in 1% sodium dodecyl sulfate and 500 ,ug/ml predigested Pronase B (50 mg/ml in H2O, heated at 37"C. for 90 minutes).

Lysis is terminated after 80 minutes at 37 C.; unlysed cells are removed by centrifugation.

Chromosomal DNA is sheared by passing the lysatethrough an 18 guage needle; it is denatured by briefly raising the pH to 12.2. After readjusting the pH to 8.5, the NaCI concentration of the lysate is brought to 3%. Denatured chromosomal DNA and proteins are extracted with redistilled phenol saturated with 3% NaCI, followed by extraction of the aqueous phase with chloroform:isoamyl alcohol (24:1). Plasmid DNA is then precipitated with two volumes of cold ethanol. After storage at -20"C. for 18 hours, DNA is pelleted, dried under vacuum, and redissolved in 10 mM Tris HCI, pH 8.0,0.1 mM EDTA.

Plasmid DNA is further purified by caesium chloride-ethidium bromide density gradient centrifugation. Caesium chloride is dissolved in the DNA solution art a ratio of 1:1 (wt.:vol.), followed by addition of 550,tzg/ml ethidium bromide. Gradients are centrifuged for approximately 40 hours atca.

100,000 x g. Plasmid DNA is removed from the gradient by needle puncture, and the ethidium bromide extracted with H2O-saturated 1-butanol. DNA is then dialyzed in 10 mM Tris HCI, pH 8.0,0.1 mM EDTA, followed by a final ethanol precipitation. Purified plasmid DNA is dissolved in 10 mM Tris HCI, pH 8.0,0.1 mM EDTA.

The method, described above, for preparing pOP 203 DNA also can be used to prepare pOV 230 DNA or other plasmid DNA. Also, it is within the skill of those in the artto vary the above conditions to pre- pare plasmid DNA.

Example 2 -- Restriction Endonuclease Digestions (a) EcoRI digestion of the pOP 203 DNA, prepared as described in Example 1, is done in a reaction mixture containing 100 mM Tris HCI, pH 7.4, 50 mM NaCI, 5 mM MgCl2, 100 ijgmlml autoclaved gelatin, 70,agm/ml DNA and 100 Units/ml EcoRI restriction endonuclease. After incubation for 60 minutes at 37 C., the reaction mixture is phenol extracted, ether extracted, and ethanol precipitated. It should be recognized that the use of another vehicle would probably require the use of a different restriction endonuclease.

(b) Taq I digestion of the pOV 230 DNA, prepared as described in Example 1, is done in a reaction mixture containing 6 mM Tris 'HCl, pH 7A, 6 mM NaCI, 6 mM MgCl2, 6 mM 2-mercaptoethanol,100,agm/ml autoclaved gelatin,40,ugm/ml DNA and 40 Units/ml Taq I restriction endonuclease. After incubation for 60 minutes at 65 C., the reaction mixture is treated as above in Example 2 (a) forthe EcoRI digestion. Other restriction endonucleases can be used so long as the cut is to the 5' side of the ovalbumin initiation codon.

Use of a different vehicle or gene would probably require the use of a different restriction endonulease.

(c) Hha I digestion of pOV 230 DNA which had been digested with Taq I, filled in using DNA polymerase, and ligated with EcoRI linkers using DNA ligase prepared as described in Example 6 (a), is done, advantageously, in a reaction mixture containing 6 mM Tris HCI, pH 7.4, 50 mM NaCI, 6 mM MgCI2, 6 mM 2-mercaptoethanol,100,agm/ml autoclaved gelatin, 60 pgmlml DNA, and 200 Units/ml Hha I restriction endonuclease. After incubation for 60 minutes at 37 C., the following components are added to the reaction mixture: Tris HCI, pH 7.4, to make the final concentration 100 mM, autocalved gelatin to maintain 100,agm/ml concentration, and 130 Units /ml of EcoRI.The reaction is incubated at 37"C. for 60 minutes and worked up as described in Example 2 (a). If linkers other than those containing an EcoRI cleavage site are used, then a different restriction endonuclease will be required.

It is within the skill of those in the art to vary the concentrations of reagents, substrates and enzymes, as well as reaction conditions to obtain the desired cleavages.

Example 3 -Alkallne Phosphatase Treatment This procedure is carried out essentially as described by Ullrich etal, (1977) Science 196: 1313-1319, with some minor modifications. Twelve Units/ml of bacterial alkaline phosphatase (BAPF, Worthington) in 20 mM Tris HCI, pH 8.0, are preincubated at70 C. for 10 minutes. One hundred ,agm/ml of EcoRl cut pOP 203 DNA, prepared as described in Example 2 (a), is then added and incubation at 70"C. continues for 15 minutes. The reaction mixture is then phenol extracted three times, ether extracted, and ethanol precipitated. This procedure is optional in the preparation of pUC 1001.

However, use of the procedure affords a higher ratio of pUC 1001 to parental pOP 203 plasmid among tetracycline resistanttransformants, thereby facilitating the recovery of pUC 1001.

Example 4 - T4 DNA Polymerase T4 DNA polymerase catalyzed fill-in of staggered ends is done in a reaction mixture containing 67 mm Tris HCI, pH 8.8,6.7 mM MgCl2, 10 mM 2-mercaptoethanol, 16.6 mM (NH4)2SO4,6.7,aM EDTA,167,agm/ml autoclaved gelatin, 0.2 mM each of dCTP, dATP, dGTP, dTTP,25,agm/ml Taq I cut pOV 230 DNA prepared as described in Example 2 (b), and 30 Units/ml T4 DNA polymerase (Bethesda Research Labs., Bethesda, Maryland). Incubation is for 15 minutes at 15 C., following which 75 ;gm/ml of tRNA is added followed by phenol extraction, ether extraction and dialysis versus 10 mM Tris HCI, pH 8.0,0.1 mM EDTA, 500 mm NaCI.The DNA is then ethanol precipitated. Other enzymes, for example, E.

coli DNA polymerase and avian myeloblastosis virus (AMV) reverse transcriptase can be used to fill in staggered ends. It is within the skill of those in the art to vary the concentrations of reagents, substrates and enzymes, as well as reaction conditions to obtain the desired filling in of staggered ends.

Example 5-Preparation of EcoRl Linkers Linker DNA is received as single stranded octamers without 5' phosphates. The 5' phosphates are added using polynucleotide kinase in a reaction mixture containing 70 mM Tris HCI, pH 7.8, 15 mM 2-mercaptoethanol, 10 mM MgCl2, 0.25 mM ATP, 40 ,agm/ml autoclaved gelatin,30,mgm/ml RI octamers (supplied as molecular recombination linkers No.

18027 by Collaborative Research, Waltham, Mass.) and 90 Units/ml polynucleotide kinase (New England Biolabs, Beverly, Mass.). The reaction mixture is incubated at 37"C. for 30 minutes, when an additional 90 Units/ml of enzyme are added and the incubation continued for another 30 minutes. The mixture is then heated to 700C. for 10 minutes and slowly cooled to 4"C, to allow annealing of doublestranded linkers.

Example 6- DNA Ligase (a) About two-thirds of the reaction mixture for ligation of synthetic EcoRI linkers to filled-in Taq I cut pOV 230, prepared as described in Example 4, consists of phosphorylated, double-stranded linkers contained in the reaction mixture described in Example 5. The final concentration of materials is 45 mM Tris HCI, pH 7.8, 10 mM 2-mercaptoethanol, 10 mM MgCl2, 15 mM dithiothreitol, 1 mM ATP, 20 ,agm/ml Taq I cut pOV 230 DNA, and 14 Units/ml of T4 DNA ligase (New England Biolabs). The reaction is incubated at 12.5 C. for approximately 16 hours and then phenol extracted, ether extracted and ethanol precipitated.

(b) In order to ligate the ovalbumin gene fragment, purified as described in Example 7, to the alkaline phosphatase treated pOP 203 DNA, prepared as described in Example 3, the reaction mixture contains 50 mM Tris - HCI, pH 7.8, 10 mm MgCl2, 20 mM dithiothreitol, 1 mM ATP, 30 ,ugm/ml pOP 203 DNA, 6 ,ugm/ml purified pOV 230 fragment, and 15 Units/ml of T4 DNA ligase.After incubation for 16 hours at 12.5"C., the reaction mixture is ethanol precipitated and the pellet dissolved in TCM (10 mM Tris HCI, pH 8.0, 10 mM CaCI2, 10mM MgCl2). It is within the skill of those in the artto vary the concentrations of reagents, substrates and enzymes, as well as reaction conditions, to obtain the desired ligations.

Example 7 -- Preparative Agarose Gel Electrophoresis Agarose is dissolved to 1% in 2x E buffer (0.08 M Tris - HCI, pH 7.8, 0.01 M NaC2H302, 0.02 M EDTA) and poured into a Bio-Rad slab gel apparatus. Samples are dissolved in 10 mM Tris - HCI, pH 8.0, 0.1 mM EDTA and the samples are run at constant power with 2x E running buffer.

After electrophoresis of the Hha I and EcoRI digested DNA, prepared as described in Example 2 (c), one lane is cut from the gel, stained with ethidium bromide (0.5 ,agm/ml) and the DNA bands visualized under ultraviolet light. The band of interest is cut from the rest of the gel and macerated before passing it through a 20 guage needle. An equal weight of extraction buffer (10 mM Tris - HCI, pH 8.0, 2 mM EDTA, 1 M NaCI) is then added and mixed with the gel. The mixture is incubated at 47"C.

for 16 hours and the agarose pelleted at 100,000 x g for 1 hour. The supernatant is then made 30 ,agm/ml in tRNA and extracted with phenol until no agarose is visible at the interface. The DNA is then ether extracted and ethanol precipitated. Gel buffers and extraction procedures can be varied by one skilled in the artto recoverthe desired DNA fragments.

Example 8 -- Transformation of E. coli One hundred twenty ml of L-broth (1% tryptone, 0.5% yeast extract, 0.5% NaCI) are inoculated with an 18 hourculture of HB101 NRRL B-11371 and grown to an optical density of 0.6 at 600 nm. Cells are washed in cold 10 mM MgSO4 and resuspended for 15 minutes in 20 ml chilled 50 mM CaCI2. Bacteria are then concentrated to one tenth of this volume in CaC12 and mixed 2:1 (v:v) with ligated DNA, prepared as described in Example 6 (b). After chilling the cell-DNA mixture for 15 minutes, it is heat shocked at 420C. for 2 minutes, then allowed to equilibrate at room temperature for ten minutes before addition of L-broth 2 1/3 times the volume of the cell-DNA suspension.Transformed cells are incubated in broth at 37"C. for one hour before inoculating selective media (L-agar plus 10 ,ug/ml tetracycline) with 200 ,ul/per plate. Plates are incu bated at 37"C. for 48 hours to allow the growth of transformants. Although the transformation proce dure is essential for the amplification of biochemi cally constructed recombinant DNA molecules, the choice of conditions for such a procedure can be changed by those skilled in the artto achieve the desired purpose.

Example 9 -- Polyacrylamide Gel Electrophoresis SDS polyacrylamide gel electrophoresis for protein separation is done as described by Laemmli, (1970) Nature227: 680-685, using the Bio-Rad slab gel apparatus. Samples are dissolved in an equal volume of sample buffer (0.0625 M Tris HCI, pH 7.8, 3% w/v SDS, 5% v/v 2-mercaptoethanol, and 20% v/v glycerol) and heated at 100 C. for 5 minutes [Moir and Brammar, (1976) Mol. Gen. Genet. 149: 87-99].

Example 10 - Immunoassays The in situ immunoassay was developed by Shalka and Shapiro, (1976) Gene 1: 65-79. Bacteria are grown on supplemented N-Z bottom agar (1% N-Z amine A, 0.5% NaCI, 0.4% casamino acids, 10 ,agm/ml thymine, 1.5% agarose) containing 25,mI/ml of ovalbumin antisera (Antibodies, Inc., Davis, California), 10 ELg/ml tetracycline and 1 mM IPTG.

After growth overnight at370C., the colonies are lysed in situ by embedding them in 0.6% agarose containing 0.5 mg/ml lysozyme (LYSF, Worthington, Freehold, N.J.), 0.1 M Tris - HCI, pH 8.0, and 10 mM EDTA. After 60 minutes at 37"C., the agarose is overlaid with 2% sarkosyl (1 ml/plate) and incubation is continued overnight at 37"C.

Ovalbumin immunoreactive material in cell extracts is determined by diffusion of the extract from a well into agarose containing ovalbumin antisera. Plates contain 1.3 /O agarose in 10 mM Tris HCI, pH 7.4, 100 mM NaCI, and 1 cLllml of ovalbumin antiserum. Wells are cut with a blunt 13 guage needle and 3 l of extract, obtained as described in Example 11, are added to each well. The plates are incubated at 37"C. overnight to allow precipitated halos to form.

Immunoreactive material is precipitated for gel analysis by adding antiserum to extracts. The mixtures are allowed to stand for 60 minutes at room temperature, and then for about 16 hours at4 C. The precipitate is centrifuged in the cold at 2000 x g for 5 minutes and washed two times with 25 mM Tris HCI, pH 7.4,137 mM NaCI and 5 mM KCI.

Example 11- Cell Extract Preparation Cells transformed as described in Example 8, are grown in L-broth containing 10 Ccg/ml tetracycline with or without 1 mM IPTG. When growth reaches the desired OD at 600 nm, cells are pelleted, washed with 0.1 M Tris HCI, pH 8.0, and frozen at-700C.

Cells are placed in a chilled mortar while frozen and ground with 2.5 times their weight of alumina at 4"C. Extraction buffer (10 mM Tris - HCI, pH7A, 10 mM MgCl2, 50 mM NaCI) is added ata ratioof2:1 volume (mls):cell weight (gms). One pg DNase (Worthington) per gram of cells is also added, fol lowed by incubation at4 C. for at least 10 minutes before removing alumina and insoluble cellular material by centrifugation.

Protein concentrations are determined by the method of Lowry et al, (1951) J. Biol. Chem. 193: 265-275, using bovine serum albumin as the stan dard.

Example 12- Purffication Of Ovalbumin The fact that ovalbumin synthesized in E. coli is secreted into the peiplasmicspace allows it to be purified relatively easily. Cells transformed, as described in Example 8, are grown in L-broth con taining 10,ag/ml tetracycline with 1mM IPTG, and harvested at late log phase. The starting material for purification is the supernatant fraction obtained when theE. coli are converted to protoplasts with lysozyme and EDTA in an osmotically protective medium [Malamy and Horecker, B. L. (1964) Biochemistry 1893-1897]. This single step removes the bulk of protein and nucleic acid from the preparation, and affords a preparation of ovalbumin which can be used as a feed supplement.

Further purification to obtain crystalline ovalbumin, for use in the baking industry or for research purposes, can be done as described by Shepherd and Montgomery, (1976) in Methods in Carbohydrate Research, Vol. VII, Whistler and BeMiller, ed., Academic Press, New York, pages 172-174. Crystalline ovalbumin is listed in the sales catalogues of various fine chemical suppliers.

Structural genes coding for eukoryotic proteins can generally be prepared using their purified messenger RNAs (mRNAs) as starting material. A complementary DNA (cDNA) copy of the mRNA is enzymatically synthesized and then enzymatically made double-stranded. This gene is then joined to a suitable cloning vehicle, usually by the poly dA:poly dTtailing procedu re [Jackson etal (1972) Proc. Nat.

Acad. Sci. 69: 2904-2909], although this is not always necessary. The vehicle containing the structural gene is then amplified in bacteria. This procedure has been used to prepare the pOV 230 plasmid, as well as plasmids containing globin genes and insulin genes.

Isolation and cloning of control elements to turn on the microbial synthesis of animal proteins in recombinant DNA molecules is relatively straightforward once an assay has been developed. The chromosomal DNA containing the desired control elements can either be cut with restriction endonucleases and directly cloned into a vector, or put on a transducing phage prior to cutting with endonuclease. The assay used to detect clones lay control elements is based on the constitutive synthesis of p-galactosidase in cells harbouringthelac operator cloned on a multicopy plasmid. Colonies of these cells turn blue on agar plates containing 5 - chloro - 4 - bromo - 3 - indolyl - p - D - galactoside; see Back man et al, Acad. Sci. 73 (1976)4174A178.

Claims (29)

1. A process for the expression of a gene coding for a high molecular weight protein of at least 10,000 daltons in a suitable vehicle, which comprises fusing the gene near transcriptional and translational initiation regions in the vehicle while maintaining the translational reading frame, and inserting the vehicle into a suitable host.

2. A process according to claim 1 wherein the vehicle is a plasmid.

3. A process according to claim 2 wherein the plasmid is pOP 203.

4. A process according to claim 2 wherein the plasmid contains an E. colilac promotor and an E.

coli p-galactosidase initiation region.

5. A process according to any preceding claim wherein the transcriptional and translational initiation regions naturally initiate mRNA synthesis and protein synthesis in a unicellular organism.

6. A process according to claim 5 wherein the transcriptional initiation region is the lac promotor, and the translational initiation region is the E. coli ss-galactosidase initiation region.

7. A process according to any preceding claim wherein the host is a unicellular organism.

8. A process according to any of claims 5 to 7 wherein the unicellular organism is a fungus.

9. A process according to any of claims 5 to 7 wherein the unicellular organism is a bacterium.

10. A process according to claim 9 wherein the bacterium is E. coli.

11. A process according to claim 10 wherein the bacterium is the E. coli K-12 derivative HB101.

12. A process according to any preceding claim wherein the gene codes for an animal protein.

13. A process according to claim 12 wherein the gene is isolated from a vertebrate.

14. A process according to claim 13 wherein the vertebrate is warm-blooded.

15. A process according to claim 14 wherein the vertebrate is a bird.

16. A process according to claim 15 wherein the bird is a chicken.

17. A process according to claim 16 wherein the protein is chicken ovalbumin.

18. A process according to any of claims 1 toll wherein the gene codes for a plant protein.

19. A process according to any of claims 1 to 11 wherein the gene is from a plant virus.

20. A process according to any of claims 1 to 11 wherein the gene is from an animal virus.

21. A process according to claim 1 substantially as described in any of the Examples.

22. A process according to claim 1 substantially as described with reference to the accompanying drawing.

23. E. coli HB101, NRRL B-11356.

24. Plasmid pUC 1001.

25. A process for constructing a plasmid in which a transcriptional initiation site and translational initiation site are fused to the structural gene coding for a high molecular weight protein, which comprises: (a) cutting a suitable cloning vehicle containing the desired transcriptional and translational initiation sites with a restriction endonuclease to obtain a first linear molecule containing the transcriptional and translational initiation sites; (b) cutting a cloned DNA molecule containing the DNA sequence coding for the desired protein to obtain a second linear molecule containing the DNA sequence coding for the desired protein; ; (c) so modifying the first and/orthe second linear DNA molecule that the translational reading frame initiated in the first linear DNA molecule is in phase with the natural initiation codon in the second linear DNA molecule when the first and second linear DNA molecules are joined together.

26. A process according to claim 25 wherein the protein is chicken ovalbumin.

27. A process according to claim 25 or claim 26 wherein the cloning vehicle is pOP 203 plasmid.

28. A process according to any of claims 25 to 27 wherein the second linear DNA molecule is obtained from pOV 230 plasmid.

29. A process for preparing chicken ovalbumin, which comprises culturing E. coli HB101 (pUC1001), NRRL B-11356, in an aqueous nutrient medium.