Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6478—Aspartic endopeptidases (3.4.23)
  • C12N9/6481—Pepsins (3.4.23.1; 3.4.23.2; 3.4.23.3)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
  • C07K1/1136—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents

Definitions

• This invention relates to the field of protein production using recombinant DNA biotechnology.
• it relates to a process for preparing chymosin from an insoluble form of a chymosin precursor produced by a host organism transformed with a vector including a gene coding for the chymosin precursor.
• proteins which may be produced in large quantities by culturing a host organism capable of expressing heterologous genetic material. Once a protein has been produced by a host organism it is usually necessary to treat the host organism in some way, in order to obtain the desired protein in a free form. In some cases, such as in the production of the interferon in E. coli a lysis or permeabilisation treatment alone may be sufficient to afford satisfactory yields. However, some proteins are produced within a host organism in the form of insoluble protein aggregates which are not susceptible to extraction by lysis or permeabilisation treatment alone. It has been reported for instance that a human insulin fusion protein produced in E. coli forms insoluble protein aggregates (see D.C. Williams et al Science Vol.
• a protein exists as a chain of amino acids linked by peptide bonds.
• the chain is folded into a thermodynamically preferred three dimensional structure, the conformation of which is maintained by relatively weak interatomic forces such as hydrogen bonding, hydrophobic interactions and charge interactions.
• a number of S-S covalent bonds may form intramolecular bridges in the polypeptide chain.
• the insoluble proteins produced by certain host organisms do not exhibit the functional activity of their natural counterparts and are therefore in general of little use as commercial products. The lack of functional activity may be due to a number of factors but it is likely that such proteins produced by transformed host organisms are formed in a conformation which differs from that of their native states.
• the altered three dimensional structure of such proteins not only leads to insolubility but also diminishes or abolishes the biological activity of the protein. It is not possible to predict whether a given protein expressed by a given host organism will be soluble or insoluble.
• the chymosin precursor proteins produced by various host organisms used were not produced in their native form.
• the methionine-prochymosin produced by E. coli is almost entirely produced as an insoluble aggregate and about 75% of the methionine-prochymosin produced in Saccharomyces cerevisiae is produced in an insoluble form.
• the proteins produced by a host organism must be solubilised and converted into their native form before the standard techniques of protein purification and cleavage may be applied.
• a process for production of chymosin in which an insoluble form of a chymosin precursor is produced by a host organism transformed with a vector including a gene coding for the chymosin precursor wherein the insoluble form of the chymosin precursor is solubilised to produce the soluble native form of the chymosin precursor prior to cleaving the soluble native form of the chymosin precursor to produce chymosin.
• the insoluble form of the chymosin precursor produced by the host organism is preprochymosin or a fusion protein including the amino acid sequence of preprochymosin, prochymosin or chymosin, for example, methionine-prochymosin or methionine-chymosin.
• Methionine-prochymosin is preferred as the chymosin precursor
• the host organism may be a host organism or the progeny of a host organism which has been transformed (using the techniques described in our published British patent application GB2100737A) with a vector including heterologous genetic material coding for the chymosin precursor.
• the host organism may be a yeast, for example, Saccharomyces cerevisiae or a bacterium, for example, E. coli, B. subtilis, B. stearothermophilis , or
• Saccharomyces cerevisiae or E. coli are the preferred host organisms.
• the expression system comprising E. coli transformed with a vector including a gene coding for the chymosin precursor, methionine-prochymosin, is especially preferred.
• insoluble' as used herein means in a form v/hich, under substantially neutral conditions (for example pH in the range 5.5 to 8.5), is substantially insoluble or is in insolubilised association with insoluble material produced on lysis of host organism cells.
• the insoluble product is either produced within the cells of the host organism in the form of insoluble relatively high molecular weight aggregates or may simply be associated with insoluble cell membrane material.
• Denaturing has the effect of abolishing the weak interatomic forces which maintain the protein in its three dimensional form causing the protein to unfold.
• the covalent bonds between adjacent atoms in the protein are left intact, including S-S bonds which maintain some of the three dimensional structure of the protein.
• the denatured state of a protein is less compact and is generally catalytically inactive.
• the denatured state is however usually soluble in the denaturing solution used. Removal of the denaturant from the solubilised proteins results in the refolding of the protein to produce the thermodynamically preferred native state of the protein.
• the renaturing is accompanied by the appearance of biological activity.
• the solubilisation involves reversibly denaturing the insoluble form of the chymosin precursor and subsequently allowing the chymosin precursor to renature, thereby producing the soluble native form of the chymosin precursor.
• the insoluble form of the chymosin. precursor is denatured in an aqueous solution comprising urea at a concentration of at least 7M and the chymosin precursor is renatured subsequently by reducing the concentration of urea in the solution below a concent ration effective to denature the chymosin precursor, to produce a soluble native form of the chymosin precursor.
• the insoluble chymosin precursor When the insoluble chymosin precursor is treated with urea the insoluble precursor is completely solubilised.
• the disulphide intramolecular bridges of the protein are however preserved and subsequently act as a nucleus for refolding.
• the urea When the urea is removed, for example by dialysis, the protein returns to a thermo dynamically stable conformation which.
• chymosin precursors In the case of chymosin precursors, is a conformation capable of being converted to active chymosin by the methods described in published British patent application. GB2100737A .
• the renatured proteins have the solubility characteristics of the native proteins.
• the insoluble form of the chymosin precursor is denatured in an aqueous solution comprising guanidine hydrochloride at a concentration of at least 6M and the chymosin precursor is renatured subsequently by reducing the concentration of guanidine hycrochloride in the solution below a concentration effective to denature the chymosin precursor, to produce a soluble native form of the chymosin precursor.
• the insoluble form of the chymosin precursor is denatured in an alkaline aqueous solution of between pH 9 and pH 11.5 and the chymosin precursor is renatured subsequently by reducing the pH of the solution below a pH effective to denature the chymosin precursor, to produce the soluble native form of the chymosin precursor.
• the alkaline aqueous solution is of pH between pH 10 and pH 11.
• Most preferred is an alkaline aqueous solution of pH from 10.5 to 10.9, preferably about 10.7.
• Treatmeat of an insoluble chymosin precursor extract with an alkaline solution as described above does not result in complete solubilisation of the chymosin precursor. Since insoluble material such as cell debris is present at all times, a number of mass transfer effects are important. It has been found that multiple extractions with alkali are more efficient than a single extraction even when large extraction volumes are used. This also has the advantage of minimising the time for which the solubilised chymosin precursor is in contact with alkali. This is of importance since there is evidence that prochymosin slowly loses activity in alkaline solutions.
• the insoluble form of the chymosin precursor is present in conjunction with debris derived from the host organism which is insoluble in the aqueous solution and wherein one or more extractions of denatured chymosin precursor are performed.
• the alkali solubilisation technique is attractive in terms of commercial exploitation.
• the methods of solubilisation in a strong denaturant such as guanidine hydrochloride or urea and solubilisation using alkali each solubilise significant percentages of the insoluble chymosin precursor which are found in extracts from host organisms.
• the insoluble form of the chymosin precursor is denatured in an aqueous solution
• the resulting solution is diluted into 10 to 50 volumes of an alkaline aqueous solution of between pH 9 and pH 11.5 and the chymosin precursor is renatured by reducing the pH of the solution below a pH effective to denature the chymosin precursor, to produce the soluble native form of the chymosin precursor.
• the solution containing the denatured chymosin precursor is diluted into an alkaline aqueous solution of pH between pH 10 and pH 11 and more preferably into an alkaline aqueous solution of pH from 10.5 to 10.9 , preferably about 10.7.
• the dilution introduces an element of physical separation between the denatured molecules, before renaturation is brought about, for example, by neutralisation of the alkaline denaturing solution.
• the dilution and resulting physical separation of the denatured molecules appears to assist their renaturation.
• the solubilisation process described immediately above leads to a recovery, in the case of methionine-prochymosin, of more than 30% compared to, for example, 10 to 20% for the multiple alkali extractions also described above.
• the insoluble form of the chymosin precursor is denatured in an aqueous solution comprising urea at a concentration of at least 7M or in a solution comprising guanidine hydrochloride at a concentration of at least 6M.
• the present invention is preferably applied to the solubilisation of insoluble methionine-prochymosin produced by a host organism transformed with a vector including a gene coding for methionine-prochymosin.
• the host organism is E. coli.
• Example 1 Some embodiments of the present invention are now described in detail by way of Examples.
• Example 1
• E. coli/pCT70 cells grown under induced conditions were suspended In three times their weight of 0.05 M tris-HC1 pH8, 1 mM EDTA, 0.233 M NaCl, 10% glycerol (v/v) containing 130 ⁇ g/ml of lysozyme and the suspension was incubated at 4° for 20 minutes.
• Sodium deoxycholate was added to a final concentration of 0.05% and 10 ijg of DNAase 1 (from bovine pancreas) was added per gram of E. coli starting material. The solution was incubated at 15°C for 30 minutes by which time the viscosity of the solution had decreased markedly.
• DNA ase 1 from bovine pancreas was added per gram of E.coli starting material.
• the solution was incubated at 15°C for 30 minutes by which time the viscosity of the solution had decreased markedly.
• the extract, obtained as described above, was centrifuged for 45 minutes at 4°C and 10000 x g.
• Example 3 An experiment was conducted in which the solubilisation of methionine-prochymosin produced by E.coli cells transformed with vector PCT70 was achieved using denatura tion with guanidine hydrochloride, followed by dilution into an alkaline solution. The preparation of the transformed cell line is described in detail in published British patent application GB2100737A.
• E. coli/ pCT70 cell debris containing insoluble methionine-prochymosin was prepared and washed as described in Example 1 above and the following manipulations were carried out at room temperature.
• the cell debris was dissolved in 3-5 volumes of buffer to final concentration of 6M guanidine HCl/0.05 M Tris pH8 , ImM EDTA, 0.1m NaCl and allowed to stand for 30 mins - 2 hrs.
• the mixture was diluted into 10-50 volumes of the above buffer at pH 10.7 lacking guanidine HCl . Dilution was effected by slow addition of the sample to the stirred diluent over a period of 10-30 minutes..
• the diluted mixture was readjusted to pH 10.7 by the addition of 1 M NaOH and allowed to stand for 10 mins - 2 hrs.
• the pH was then adjusted to 8 by the addition of 1N

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• 1983
  • 1983-06-07 AU AU16073/83A patent/AU553017B2/en not_active Ceased
  • 1983-06-07 GB GB08401585A patent/GB2129810B/en not_active Expired
  • 1983-06-07 WO PCT/GB1983/000152 patent/WO1983004418A1/en not_active Ceased
  • 1983-06-07 EP EP83901829A patent/EP0112849B1/en not_active Expired
  • 1983-06-07 NZ NZ204477A patent/NZ204477A/en unknown
  • 1983-06-07 IE IE1341/83A patent/IE55163B1/en not_active IP Right Cessation
  • 1983-06-07 JP JP58501900A patent/JPS59500996A/ja active Granted
  • 1983-06-07 DE DE8383901829T patent/DE3373676D1/de not_active Expired
• 1984
  • 1984-02-03 DK DK49984A patent/DK49984A/da not_active Application Discontinuation
  • 1984-02-06 FI FI840468A patent/FI78318C/fi not_active IP Right Cessation

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