CA1337283C - Method for the selective cleavage of fusion proteins - Google Patents
Method for the selective cleavage of fusion proteinsInfo
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- CA1337283C CA1337283C CA000583545A CA583545A CA1337283C CA 1337283 C CA1337283 C CA 1337283C CA 000583545 A CA000583545 A CA 000583545A CA 583545 A CA583545 A CA 583545A CA 1337283 C CA1337283 C CA 1337283C
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
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Abstract
The invention relates to a method for the preparation of polypeptides or proteins by enzymatic cleavage of the oligo-or polyglycine sequence of a fusion protein using an endo-protease.
Description
-I -Description A method for the selective cleavage of fusion proteins The invention relates to a method for the preparation of polypeptides or proteins by enzymatic cleavage of a fusion protein.
The increasing importance of recombinant DNA technology for obtaining polypeptides and proteins requires the develop-ment of new methods for enriching and purifying the products, which are appropriate for the altered starting materials.
At present, a large number of proteins is synthesized in the microorganism as fusion protein, i.e. the sequence of a foreign protein is placed in front of the amino acid sequence of the desired polypeptide (F.AØ Harston, Biochem.
J. 240 (1986) 1-12). In general, the fusion proteins pre-cipitate in the cell, because they are sparingly soluble or insoluble, as what are called inclusion bodies and are thus - protected from proteolytic degradation. This ensures high yields and ease of isolation of this primary gene product.
However, in order to obtain the desired polypeptide it has to be separated out of the fusion protein by enzymatic or chemical cleavage. Chemical methods are often used for cleavage, because it is most straightforward to make them appropriate for the sparingly soluble nature of the fusion protein. Incomplete cleavage or formation of byproducts by irreversible derivatization of amino acid side-chains are, however, observed with virtually all chemical methods.
There is also always a danger of non-specific degradation of polypeptide chains (K.-K. Han et al., Int. J. Biochem.
15, (1983) 875-884). Moreover, the use of chemical methods is restricted because the specificity of the cleavage is determined predominantly by a single amino acid which, in the nature of things, is often also present in the desired poLypeptide.
Enzymatic fragmentations can be carried out under consider-ably milder conditions. However, general difficulties arise here due to the fact that the sparingly soluble fusion protein must be dissolved and maintained in solution using detergents, urea or guanidine hydrochloride, that is to say conditions under which enzymes are often inactivated. It is often impossible to use proteases which recognize only a single specific amino acid because this amino acid is also present in the desired protein. On the other hand, the availability of proteases which recognize and cleave only very specific, rare sequences of several amino acids is low.
Thus, it is necessary to find a method of cleavage specific for each product. Hence, it is additionally desirable to have a universally applicable cleavage method which cleaves only at very particular, rare amino acid sequences without damaging the protein and which can also be applied to sparingly soluble fusion proteins.
Lysostaphin is disclosed in the literature as an enzyme - which degrades cell walls and is secreted by Staphylococcus simulans (NRRL B-2628; Sloan et al., Int. J. of Systematic Bacteriology, Vol. 32, No. 2 (1982) 170-174) into the medium. This enzyme lyses virtually all known Staphylo-coccus species but no other bacterial species. It has hitherto been assumed that lysostaphin endoprotease only cleaves, very selectively, the polyglycine bridges in the murein sacculus of the Staphylococci (Iversen and Grov, Eur. J. Biochem. 38 (1973) 293-300). Additionally disclosed have been transpeptidization experiments with lysostaphin catalysis using short synthetic glycylpeptides lG. L. Sloan et al., Biochem. J. 167 (1977) 293-296). ~e have now found, surprisingly, that lysostaphin endoprotease also cleaves fusion proteins having an oligo- or poly-glycine sequence. It is apparently unnecessary for the selectivity of the cleavage that this sequence be bound into the specific steric relationships of a bacterial cell wall.
~his makes it possible to eliminate specifically the desired protein from a fusion protein under mild conditions.
~ he present invention relates to a method for the prepara-tion of polypeptides or proteins by enzymatic cleavage, ~hich comprises cleavage of a fusion protein having the sequence (Y1 ~ Ym) ~ (Gly)p+q - (X1 -Xn) ~here (Y1...Ym)-(Gly)p represents the sequence ~hich is to be eliminated and (Gly)q-(X1...Xn) represents the polypeptide or protein, X and r denote, independently of one another, natural amino acids, m and n denote numbers greater than 1, p and q each denotes a number greater than O and p ~ q together denote a natural number bet~een ~ and 100, and if Ym represents Gly it is possible for p + q also to be < 2 and p to be 0, and if X1 represents Gly it is possible for p + q also to be < 2 and q 2G to be 0, uith an endoprotease specific for oligo- or polyglycine sequences, and subjecting the polypeptide or protein ~hich is liberated by this to further chemical or enzymatic treatment ~here appropriate, or directly processing it further.
Dependent on the value of the indices p and/or q, the sequence which is to be eliminated and/or the polypeptide or protein is optionally su~jected to further enzymatic cleavages.
(r1 --- Ym) is a natural or artificial protein sequence as customarily employed for the preparation of fusion proteins. Suitable examples are B-galactosidase, en~ymes of tryptophan metabolism or parts of these protein molecules uhich, in general, result in insolub(e products, as ~ell as polypeptide sequences ~hich facilitate rapid enrichment of a soluble fermentation product (for example antibodies).
(X1 -. Xn) represents a pharmacologically active poly-peptide or protein or represents a higher molecular weight precursor from ~hich the desired biologically active form is obtained by further processing such as folding, ~ith the production of correct disulfide bridges, and/or specific cleavage of the polypeptide chains. One example of this ~ould be preproinsulin, from ~hich insulin is produced.
The residues X and Y represent, independently of one another, naturally occurring amino acids (see, for example, Schro'der, Lubke "The Peptides" Vol. I, Ne~ York 1965, Pages 137-270 and Houben-~eyl "Methoden der organischen Chemie" (Methods of Organic Chemistry) Vol. 15/1 and 2 (Synthesis of Peptides), Georg Thieme Verlag Stuttgart 1974, Annex). The follo~ing may be particularly mentioned: Gly, Ala, Ser, Thr, Val, Leu, Ile, Asp, Asn, Glu, Gln, Cys, Met, Arg, Lys, Hyl, Orn, Cit, Tyr, Phe, Trp, His, Pro and Hyp.
An endoprotease specific for oligo- and polyglycine se-quences is ~o be understood to be, in particular, lysosta-phin tmanufacturer: (R)SIGMA Chemie GmbH, Deisenhofen; cf.
also Recsei et al., Proc. Natl. Acad. Sci. USA, Vol. 84, (1987) 1127-1131).
The desired product (X1 ... Xn) is connected to a protein (r1 ... Ym) by any desired glycine sequence (Gly)p + q.
Preferred fusion proteins are those in ~hich p ~ q together denotes a natural number bet~een 2 and 100. A larger number of consecutive glycine residues may in this connec-tion have a beneficial effect on the reaction rate, becausethe cleavage site is more accessib(e. Ho~ever, the same effect is also achieved ~hen short oligoglycine sequences are flanked by one or more other amino acids which do not sterically hinder the enzyme during cleavage. Accordingly, the length of the connecting piece should be made appro-priate for the structural properties of the fusion partners.
_ 5 _ l 3 3 7 2 8 3 The cleavage conditions can be varied vithin a very ~ide range and thus made appropriate for the properties of the fusion protein. Thus, it is possible for the enzyme/
substrate ratio to be, for example, betveen 1:1 and 1:1,000,000, and for the reaction to be carried out in a pH range from 6 to 9, preferably in the range 7 to 8, preferably at a temperature of 20-60C, especially of 32-42C. It is also possible, vhere appropriate, depend-ing on the degree of the sparing solubility of the fusion protein, to add an auxiliary, for example urea, detergents or guanidine hydrochloride, vhich keeps the fusion protein in solution. Although the cleavage takes place most rapidly vhen virtually complete solution of the fusion protein is possible vithout addition of denaturing agents, the lysostaphin endoprotease is not inactivated by the presence of urea, merely the enz~matic reaction is slo~ed dovn. This fact can be utilized for the cleavage of particularly sparingly soluble fusion proteins. On the other hand, it is also possible in principle to carry out successfully, ~ith a corresponding increase in the reac-tion time, fragmentations of merely suspended inclusion bodies. In the case of solutions of the fusion protein -vith or vithout denaturing agent - it is also possible to use advantageously the lysostaphin endoprotease in carrier-bound for~ (immobilized en~yme) and recover it for reuse. Examples of suitable enzyme carriers are in-organic carriers such as aluminun silicates as vell as polymeric organic carriers such as agaroses, for example (R)Affi-Gel 1û (from Bio Rad), celluloses, modified poly-30 acrylamide gels vhich have amino or hydroxyl groups, orelse organic copolymers of acrylamide, methacrylates or methacrylamide and maleic anhydride. The preparation of appropriate carrier-bound enzymes is described, for example, in Hal~achs et al., Biotechnology and 8io-35 engineering XIX (1977) 1667-1677 (immobili~ation on alu-minum silicate) or German Patent 2,927,534 (Published August 1, 1981)(immobilization on cellulose).
The method according to the invention is not only suitable for the selective cleavage of fusion proteins but also applicable generally to appropriate polypeptides.
The examples ~hich follo~ serve to illustrate the present invention but without intending to restrict it to them.
Of these, Examples 3 and 4 are intended to demonstrate that the fusion protein cleavages obtained in Examples 1 10 and 2 are attributable to the incorporation of a polygly-cine sequence and not to a degradation of (X1 ... Xn) or (Y1 ... Ym)~
Example 1 15 CCeavage of a polyglycine-containing fusion protein A construction in ~hich a segment of B-galactosidase is linked via a (Gly)1g-peptide and a synthetic hexapeptide to proinsulin is used. Proinsulin forms the carboxyl end 20 of the fusion protein. The protein is enriched to the extent of about 40%.
This protein is dissolved at a concentration of 20 mg/ml in buffer (8 M urea; 50 mM Tris/HCl; pH 7.5) and adjusted, 25 by slo~ dilution ~ith S0 mM Tris/HCl, pH 7.5 and 8 M urea, pH 7.5, to various urea concentrations (see Table 1) and a protein concentration of 2 mg/ml or 10 mg/ml. Lysostaphin ((R)SIGMA) is added in the enzyme/substrate ratio of 1:100 or 1:1000 to the solutions, which are slightly cloudy in 30 each case and are maintained at 37C. Samples are taken at defined times and analyzed by SDS electrophoresis. The selective degradation of the fusion protein at the poly-glycine sequence is evident from the decrease in the fusion protein band and the formation of a ne~ band for the galac-35 tosidase fragment having a molecular ~eight lo~er by about10,000 Dalton.
Table 1 Protein Enzyme/ Urea Reaction (> 95%
concentration substrate concentration decrease in the ratio fusion protein band) (mg/ml) (M) (Hours) 2 1 : 100 4 > 20 2 1 : 100 3 20 10 2 1 : 100 2 5 2 1 : 100 1 2 1 : 100 2 20 Table 1 lists the times after which the area, evaluated by densitometry of the SDS electrophoresis, for the original fusion band has fallen below 5%. The values obtained are bet~een 1 and 20 hours depending on the urea concentration and enzyme/substrate ratio. The presence of a reducing agent, for example dithioerythritol (DTE, Cleland's reagent), which is advantageous for dissolving the fusion protein, - has no significant effect on the activity of the enzyme.
Example 2 Z5 Cleavage of a fusion protein in suspension The same construction as in Example 1 is used, but not in isolated, dried form. On the contrary, a suspension as is customarily produced when obtaining the inclusion bodies, and which contains about 50 g/l fusion protein, at about 200 g/l dry matter (90% protein), is used. 10 ml of this suspension are diluted to 200 ml with 50 mM Tris/HCl, pH 7.5, and 20 mg of lysostaphin ((R)SIGMA) are added.
After 20 hours at 37C, SDS gel electrophoresis shows only small remaining traces of unfragmented fusion protein.
Example 3 Incubation of proinsulin with lysostaphin Proinsulin (human) is dissolved at a concentration of 1 mg/ml in 50 mM Tris/HCl, pH 7.5, and lysostaphin is added in the enzyme/substrate ratio 1:100. The solution is maintained at 37C. Samples are taken after 1, 3, 5 and 20 hours and analyzed by reversed phase HPLC. This reveals no evidence of degradation of the proinsulin by lysostaphin.
Example 4 Attempt at cleavage of a fusion protein without a poly-glycine sequence A construction in which a segment of 3-galactosidase is linked via a synthetic hexapeptide to proinsulin is used.
Proinsulin forms the carboxyl end of the fusion protein.
The fusion protein is enriched to the extent of about 80%.
This protein is dissolved at a concentration of 20 mg/ml in buffer (8 M urea; 50 mM Tris/HCl; pH 7.5) and adjusted to a protein concentration of 2 mg/ml by slow dilution with 50 mM Tris/HCl, pH 7.5. Lysostaphin ((R)SIGMA) in the enzyme/substrate ratio 1:100 is added to the slightly cloudy solution, which is maintained at 37C. Analysis of samples by SDS electrophoresis shows no degradation of the fusion protein even after 20 hours.
The increasing importance of recombinant DNA technology for obtaining polypeptides and proteins requires the develop-ment of new methods for enriching and purifying the products, which are appropriate for the altered starting materials.
At present, a large number of proteins is synthesized in the microorganism as fusion protein, i.e. the sequence of a foreign protein is placed in front of the amino acid sequence of the desired polypeptide (F.AØ Harston, Biochem.
J. 240 (1986) 1-12). In general, the fusion proteins pre-cipitate in the cell, because they are sparingly soluble or insoluble, as what are called inclusion bodies and are thus - protected from proteolytic degradation. This ensures high yields and ease of isolation of this primary gene product.
However, in order to obtain the desired polypeptide it has to be separated out of the fusion protein by enzymatic or chemical cleavage. Chemical methods are often used for cleavage, because it is most straightforward to make them appropriate for the sparingly soluble nature of the fusion protein. Incomplete cleavage or formation of byproducts by irreversible derivatization of amino acid side-chains are, however, observed with virtually all chemical methods.
There is also always a danger of non-specific degradation of polypeptide chains (K.-K. Han et al., Int. J. Biochem.
15, (1983) 875-884). Moreover, the use of chemical methods is restricted because the specificity of the cleavage is determined predominantly by a single amino acid which, in the nature of things, is often also present in the desired poLypeptide.
Enzymatic fragmentations can be carried out under consider-ably milder conditions. However, general difficulties arise here due to the fact that the sparingly soluble fusion protein must be dissolved and maintained in solution using detergents, urea or guanidine hydrochloride, that is to say conditions under which enzymes are often inactivated. It is often impossible to use proteases which recognize only a single specific amino acid because this amino acid is also present in the desired protein. On the other hand, the availability of proteases which recognize and cleave only very specific, rare sequences of several amino acids is low.
Thus, it is necessary to find a method of cleavage specific for each product. Hence, it is additionally desirable to have a universally applicable cleavage method which cleaves only at very particular, rare amino acid sequences without damaging the protein and which can also be applied to sparingly soluble fusion proteins.
Lysostaphin is disclosed in the literature as an enzyme - which degrades cell walls and is secreted by Staphylococcus simulans (NRRL B-2628; Sloan et al., Int. J. of Systematic Bacteriology, Vol. 32, No. 2 (1982) 170-174) into the medium. This enzyme lyses virtually all known Staphylo-coccus species but no other bacterial species. It has hitherto been assumed that lysostaphin endoprotease only cleaves, very selectively, the polyglycine bridges in the murein sacculus of the Staphylococci (Iversen and Grov, Eur. J. Biochem. 38 (1973) 293-300). Additionally disclosed have been transpeptidization experiments with lysostaphin catalysis using short synthetic glycylpeptides lG. L. Sloan et al., Biochem. J. 167 (1977) 293-296). ~e have now found, surprisingly, that lysostaphin endoprotease also cleaves fusion proteins having an oligo- or poly-glycine sequence. It is apparently unnecessary for the selectivity of the cleavage that this sequence be bound into the specific steric relationships of a bacterial cell wall.
~his makes it possible to eliminate specifically the desired protein from a fusion protein under mild conditions.
~ he present invention relates to a method for the prepara-tion of polypeptides or proteins by enzymatic cleavage, ~hich comprises cleavage of a fusion protein having the sequence (Y1 ~ Ym) ~ (Gly)p+q - (X1 -Xn) ~here (Y1...Ym)-(Gly)p represents the sequence ~hich is to be eliminated and (Gly)q-(X1...Xn) represents the polypeptide or protein, X and r denote, independently of one another, natural amino acids, m and n denote numbers greater than 1, p and q each denotes a number greater than O and p ~ q together denote a natural number bet~een ~ and 100, and if Ym represents Gly it is possible for p + q also to be < 2 and p to be 0, and if X1 represents Gly it is possible for p + q also to be < 2 and q 2G to be 0, uith an endoprotease specific for oligo- or polyglycine sequences, and subjecting the polypeptide or protein ~hich is liberated by this to further chemical or enzymatic treatment ~here appropriate, or directly processing it further.
Dependent on the value of the indices p and/or q, the sequence which is to be eliminated and/or the polypeptide or protein is optionally su~jected to further enzymatic cleavages.
(r1 --- Ym) is a natural or artificial protein sequence as customarily employed for the preparation of fusion proteins. Suitable examples are B-galactosidase, en~ymes of tryptophan metabolism or parts of these protein molecules uhich, in general, result in insolub(e products, as ~ell as polypeptide sequences ~hich facilitate rapid enrichment of a soluble fermentation product (for example antibodies).
(X1 -. Xn) represents a pharmacologically active poly-peptide or protein or represents a higher molecular weight precursor from ~hich the desired biologically active form is obtained by further processing such as folding, ~ith the production of correct disulfide bridges, and/or specific cleavage of the polypeptide chains. One example of this ~ould be preproinsulin, from ~hich insulin is produced.
The residues X and Y represent, independently of one another, naturally occurring amino acids (see, for example, Schro'der, Lubke "The Peptides" Vol. I, Ne~ York 1965, Pages 137-270 and Houben-~eyl "Methoden der organischen Chemie" (Methods of Organic Chemistry) Vol. 15/1 and 2 (Synthesis of Peptides), Georg Thieme Verlag Stuttgart 1974, Annex). The follo~ing may be particularly mentioned: Gly, Ala, Ser, Thr, Val, Leu, Ile, Asp, Asn, Glu, Gln, Cys, Met, Arg, Lys, Hyl, Orn, Cit, Tyr, Phe, Trp, His, Pro and Hyp.
An endoprotease specific for oligo- and polyglycine se-quences is ~o be understood to be, in particular, lysosta-phin tmanufacturer: (R)SIGMA Chemie GmbH, Deisenhofen; cf.
also Recsei et al., Proc. Natl. Acad. Sci. USA, Vol. 84, (1987) 1127-1131).
The desired product (X1 ... Xn) is connected to a protein (r1 ... Ym) by any desired glycine sequence (Gly)p + q.
Preferred fusion proteins are those in ~hich p ~ q together denotes a natural number bet~een 2 and 100. A larger number of consecutive glycine residues may in this connec-tion have a beneficial effect on the reaction rate, becausethe cleavage site is more accessib(e. Ho~ever, the same effect is also achieved ~hen short oligoglycine sequences are flanked by one or more other amino acids which do not sterically hinder the enzyme during cleavage. Accordingly, the length of the connecting piece should be made appro-priate for the structural properties of the fusion partners.
_ 5 _ l 3 3 7 2 8 3 The cleavage conditions can be varied vithin a very ~ide range and thus made appropriate for the properties of the fusion protein. Thus, it is possible for the enzyme/
substrate ratio to be, for example, betveen 1:1 and 1:1,000,000, and for the reaction to be carried out in a pH range from 6 to 9, preferably in the range 7 to 8, preferably at a temperature of 20-60C, especially of 32-42C. It is also possible, vhere appropriate, depend-ing on the degree of the sparing solubility of the fusion protein, to add an auxiliary, for example urea, detergents or guanidine hydrochloride, vhich keeps the fusion protein in solution. Although the cleavage takes place most rapidly vhen virtually complete solution of the fusion protein is possible vithout addition of denaturing agents, the lysostaphin endoprotease is not inactivated by the presence of urea, merely the enz~matic reaction is slo~ed dovn. This fact can be utilized for the cleavage of particularly sparingly soluble fusion proteins. On the other hand, it is also possible in principle to carry out successfully, ~ith a corresponding increase in the reac-tion time, fragmentations of merely suspended inclusion bodies. In the case of solutions of the fusion protein -vith or vithout denaturing agent - it is also possible to use advantageously the lysostaphin endoprotease in carrier-bound for~ (immobilized en~yme) and recover it for reuse. Examples of suitable enzyme carriers are in-organic carriers such as aluminun silicates as vell as polymeric organic carriers such as agaroses, for example (R)Affi-Gel 1û (from Bio Rad), celluloses, modified poly-30 acrylamide gels vhich have amino or hydroxyl groups, orelse organic copolymers of acrylamide, methacrylates or methacrylamide and maleic anhydride. The preparation of appropriate carrier-bound enzymes is described, for example, in Hal~achs et al., Biotechnology and 8io-35 engineering XIX (1977) 1667-1677 (immobili~ation on alu-minum silicate) or German Patent 2,927,534 (Published August 1, 1981)(immobilization on cellulose).
The method according to the invention is not only suitable for the selective cleavage of fusion proteins but also applicable generally to appropriate polypeptides.
The examples ~hich follo~ serve to illustrate the present invention but without intending to restrict it to them.
Of these, Examples 3 and 4 are intended to demonstrate that the fusion protein cleavages obtained in Examples 1 10 and 2 are attributable to the incorporation of a polygly-cine sequence and not to a degradation of (X1 ... Xn) or (Y1 ... Ym)~
Example 1 15 CCeavage of a polyglycine-containing fusion protein A construction in ~hich a segment of B-galactosidase is linked via a (Gly)1g-peptide and a synthetic hexapeptide to proinsulin is used. Proinsulin forms the carboxyl end 20 of the fusion protein. The protein is enriched to the extent of about 40%.
This protein is dissolved at a concentration of 20 mg/ml in buffer (8 M urea; 50 mM Tris/HCl; pH 7.5) and adjusted, 25 by slo~ dilution ~ith S0 mM Tris/HCl, pH 7.5 and 8 M urea, pH 7.5, to various urea concentrations (see Table 1) and a protein concentration of 2 mg/ml or 10 mg/ml. Lysostaphin ((R)SIGMA) is added in the enzyme/substrate ratio of 1:100 or 1:1000 to the solutions, which are slightly cloudy in 30 each case and are maintained at 37C. Samples are taken at defined times and analyzed by SDS electrophoresis. The selective degradation of the fusion protein at the poly-glycine sequence is evident from the decrease in the fusion protein band and the formation of a ne~ band for the galac-35 tosidase fragment having a molecular ~eight lo~er by about10,000 Dalton.
Table 1 Protein Enzyme/ Urea Reaction (> 95%
concentration substrate concentration decrease in the ratio fusion protein band) (mg/ml) (M) (Hours) 2 1 : 100 4 > 20 2 1 : 100 3 20 10 2 1 : 100 2 5 2 1 : 100 1 2 1 : 100 2 20 Table 1 lists the times after which the area, evaluated by densitometry of the SDS electrophoresis, for the original fusion band has fallen below 5%. The values obtained are bet~een 1 and 20 hours depending on the urea concentration and enzyme/substrate ratio. The presence of a reducing agent, for example dithioerythritol (DTE, Cleland's reagent), which is advantageous for dissolving the fusion protein, - has no significant effect on the activity of the enzyme.
Example 2 Z5 Cleavage of a fusion protein in suspension The same construction as in Example 1 is used, but not in isolated, dried form. On the contrary, a suspension as is customarily produced when obtaining the inclusion bodies, and which contains about 50 g/l fusion protein, at about 200 g/l dry matter (90% protein), is used. 10 ml of this suspension are diluted to 200 ml with 50 mM Tris/HCl, pH 7.5, and 20 mg of lysostaphin ((R)SIGMA) are added.
After 20 hours at 37C, SDS gel electrophoresis shows only small remaining traces of unfragmented fusion protein.
Example 3 Incubation of proinsulin with lysostaphin Proinsulin (human) is dissolved at a concentration of 1 mg/ml in 50 mM Tris/HCl, pH 7.5, and lysostaphin is added in the enzyme/substrate ratio 1:100. The solution is maintained at 37C. Samples are taken after 1, 3, 5 and 20 hours and analyzed by reversed phase HPLC. This reveals no evidence of degradation of the proinsulin by lysostaphin.
Example 4 Attempt at cleavage of a fusion protein without a poly-glycine sequence A construction in which a segment of 3-galactosidase is linked via a synthetic hexapeptide to proinsulin is used.
Proinsulin forms the carboxyl end of the fusion protein.
The fusion protein is enriched to the extent of about 80%.
This protein is dissolved at a concentration of 20 mg/ml in buffer (8 M urea; 50 mM Tris/HCl; pH 7.5) and adjusted to a protein concentration of 2 mg/ml by slow dilution with 50 mM Tris/HCl, pH 7.5. Lysostaphin ((R)SIGMA) in the enzyme/substrate ratio 1:100 is added to the slightly cloudy solution, which is maintained at 37C. Analysis of samples by SDS electrophoresis shows no degradation of the fusion protein even after 20 hours.
Claims (5)
1. A method for the preparation of proinsulin by enzymatic cleavage, which comprises forming a fusion protein having the sequence (Y1 ... Ym) - (Gly)p+q-(X) where (Y1...Ym)-(Gly)p represents the sequence which is to be eliminated and (Gly)q-(X) represents proinsulin, X and Y denote, independently of one another, natural amino acids, m is greater than 1, p and q each denotes a number greater than 0 and p + q together denote a natural number between 2 and 100, and if Ym represents Gly it is possible for p + q also to be < 2 and p to be 0, and, cleaving the fusion protein with lysostaphin.
2. The method as claimed in claim 1, wherein p + q together denote a whole number between 2 and 30.
3. The method as claimed in claim 2, wherein p + q together denote a whole number between 2 and 18.
4. The method as claimed in claim 1, wherein betagalactosidase is employed as the sequence to be eliminated.
5. The method as claimed in claim 4, wherein a segment of betagalactosidase is linked to proinsulin by a (Gly)18 peptide.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19873739347 DE3739347A1 (en) | 1987-11-20 | 1987-11-20 | METHOD FOR SELECTIVE CLEAVAGE OF FUSION PROTEINS |
| DEP3739347.2 | 1987-11-20 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1337283C true CA1337283C (en) | 1995-10-10 |
Family
ID=6340882
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000583545A Expired - Lifetime CA1337283C (en) | 1987-11-20 | 1988-11-18 | Method for the selective cleavage of fusion proteins |
Country Status (19)
| Country | Link |
|---|---|
| EP (1) | EP0316748B1 (en) |
| JP (1) | JPH01160496A (en) |
| KR (1) | KR890008166A (en) |
| CN (1) | CN1033184A (en) |
| AT (1) | ATE78873T1 (en) |
| AU (1) | AU618035B2 (en) |
| CA (1) | CA1337283C (en) |
| DE (2) | DE3739347A1 (en) |
| DK (1) | DK645588A (en) |
| ES (1) | ES2051816T3 (en) |
| FI (1) | FI885328L (en) |
| GR (1) | GR3006045T3 (en) |
| HU (1) | HU204894B (en) |
| IE (1) | IE62228B1 (en) |
| IL (1) | IL88413A (en) |
| NO (1) | NO173194C (en) |
| NZ (1) | NZ226998A (en) |
| PT (1) | PT89019B (en) |
| ZA (1) | ZA888650B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5780285A (en) * | 1995-03-03 | 1998-07-14 | Genentech, Inc. | Subtilisin variants capable of cleaving substrates containing dibasic residues |
| US5837516A (en) * | 1995-03-03 | 1998-11-17 | Genentech, Inc. | Subtilisin variants capable of cleaving substrates containing basic residues |
| DK0821006T3 (en) | 1996-07-26 | 2004-08-16 | Aventis Pharma Gmbh | Insulin derivatives with increased zinc binding |
| US6265204B1 (en) | 1997-01-17 | 2001-07-24 | Genencor International, Inc. | DNA sequences, vectors, and fusion polypeptides for secretion of polypeptides in filamentous fungi |
| DE19825447A1 (en) | 1998-06-06 | 1999-12-09 | Hoechst Marion Roussel De Gmbh | New insulin analogues with increased zinc formation |
| EP1494706A4 (en) * | 2002-03-26 | 2006-10-25 | Biosynexus Inc | Antimicrobial polymer conjugates |
| CN101717449B (en) * | 2008-10-09 | 2013-06-19 | 重庆富进生物医药有限公司 | Recombinant TRAIL-Fc fusion protein as well as preparation and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3523701A1 (en) * | 1985-07-03 | 1987-01-08 | Bayer Ag | PROCESS FOR PRODUCING PROTEINS AND POLYPEPTIDES |
-
1987
- 1987-11-20 DE DE19873739347 patent/DE3739347A1/en not_active Withdrawn
-
1988
- 1988-11-10 ES ES88118673T patent/ES2051816T3/en not_active Expired - Lifetime
- 1988-11-10 AT AT88118673T patent/ATE78873T1/en not_active IP Right Cessation
- 1988-11-10 DE DE8888118673T patent/DE3873273D1/en not_active Expired - Lifetime
- 1988-11-10 EP EP88118673A patent/EP0316748B1/en not_active Expired - Lifetime
- 1988-11-17 FI FI885328A patent/FI885328L/en not_active Application Discontinuation
- 1988-11-17 PT PT89019A patent/PT89019B/en not_active IP Right Cessation
- 1988-11-18 IE IE346288A patent/IE62228B1/en not_active IP Right Cessation
- 1988-11-18 NO NO885154A patent/NO173194C/en unknown
- 1988-11-18 AU AU25695/88A patent/AU618035B2/en not_active Expired
- 1988-11-18 HU HU885958A patent/HU204894B/en unknown
- 1988-11-18 NZ NZ226998A patent/NZ226998A/en unknown
- 1988-11-18 CA CA000583545A patent/CA1337283C/en not_active Expired - Lifetime
- 1988-11-18 ZA ZA888650A patent/ZA888650B/en unknown
- 1988-11-18 KR KR1019880015202A patent/KR890008166A/en not_active Ceased
- 1988-11-18 JP JP63290349A patent/JPH01160496A/en active Pending
- 1988-11-18 DK DK645588A patent/DK645588A/en not_active Application Discontinuation
- 1988-11-18 IL IL88413A patent/IL88413A/en not_active IP Right Cessation
- 1988-11-19 CN CN88107955A patent/CN1033184A/en active Pending
-
1992
- 1992-10-22 GR GR920402186T patent/GR3006045T3/el unknown
Also Published As
| Publication number | Publication date |
|---|---|
| NO173194C (en) | 1993-11-10 |
| PT89019A (en) | 1988-12-01 |
| CN1033184A (en) | 1989-05-31 |
| PT89019B (en) | 1993-02-26 |
| AU618035B2 (en) | 1991-12-12 |
| ATE78873T1 (en) | 1992-08-15 |
| FI885328A7 (en) | 1989-05-21 |
| EP0316748B1 (en) | 1992-07-29 |
| DE3739347A1 (en) | 1989-06-01 |
| KR890008166A (en) | 1989-07-10 |
| EP0316748A2 (en) | 1989-05-24 |
| EP0316748A3 (en) | 1990-08-29 |
| ZA888650B (en) | 1989-07-26 |
| NZ226998A (en) | 1991-06-25 |
| DK645588A (en) | 1989-05-21 |
| HU204894B (en) | 1992-02-28 |
| IE62228B1 (en) | 1995-01-11 |
| IE883462L (en) | 1989-05-20 |
| AU2569588A (en) | 1989-06-29 |
| DE3873273D1 (en) | 1992-09-03 |
| IL88413A (en) | 1993-07-08 |
| NO173194B (en) | 1993-08-02 |
| ES2051816T3 (en) | 1994-07-01 |
| IL88413A0 (en) | 1989-06-30 |
| HUT50514A (en) | 1990-02-28 |
| NO885154D0 (en) | 1988-11-18 |
| GR3006045T3 (en) | 1993-06-21 |
| DK645588D0 (en) | 1988-11-18 |
| FI885328L (en) | 1989-05-21 |
| FI885328A0 (en) | 1988-11-17 |
| JPH01160496A (en) | 1989-06-23 |
| NO885154L (en) | 1989-05-22 |
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