WO1995020039A2 - Customized proteases with altered transacylation activity - Google Patents
Customized proteases with altered transacylation activity Download PDFInfo
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- WO1995020039A2 WO1995020039A2 PCT/US1995/006682 US9506682W WO9520039A2 WO 1995020039 A2 WO1995020039 A2 WO 1995020039A2 US 9506682 W US9506682 W US 9506682W WO 9520039 A2 WO9520039 A2 WO 9520039A2
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- carboxypeptidase
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- 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
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
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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
- C07K14/60—Growth-hormone releasing factors (GH-RF) (Somatoliberin)
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- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- 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)
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- C—CHEMISTRY; METALLURGY
- 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
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Definitions
- transpeptidation can be very effective under some circumstances, it is limited to substrates for which a natural protease exists and which exhibits specificity for a peptide bond close to the C-terminus.
- mutant protease enzymes capable of performing heretofor unknown N- or C-terminal modifications as well as peptide chain elongation with a variety of substrates, especially those substrates that are not reactive with the
- the invention provides customized proteases (i.e., mutant proteases), methods of making customized proteases, as well as methods of using customized proteases.
- Customized proteases are derived from known proteases such as endoproteases, exoproteases, serine proteases and cysteine proteases.
- a customized protease is a modified version of a known protease designed to provide a protease that is capable of transacylating a preselected substrate with a preselected nucleophile in a transacylation reaction not substantially catalyzed by the known protease.
- the mutant or customized protease can also exhibit improved or enhanced yields of
- the preferred preselected substrates are peptides having an acidic or basic amino acid at the penultimate position.
- the preferred preselected substrates are peptides having an acidic or basic amino acid at the penultimate position.
- nucleophiles are amino acids and amino acid derivatives such as amino acid esters and amino acid amides.
- the invention also provides methods for preparing a customized protease. These methods can involve site specific mutagenesis or random mutagenesis. Site specific mutagenesis can involve replacing a selected amino acid in the active site with a selected amino acid or by replacing the selected active site amino acid with any one of the 20 amino acids randomly. Random mutagenesis can involve replacing any amino acid of the active site with any of the other 19 amino acids.
- One method of the invention involves providing a DNA sequence that encodes the known protease
- the codon for the amino acid in the active site is deleted using restriction enzymes and the deleted codon is replaced with an oligonucleotide encoding a different amino acid residue.
- Another method of the invention involves modifying a DNA sequence encoding the known protease by inserting stop codons and/or a restriction enzyme recognition site at targeted sites to form a modified DNA sequence encoding an inactive protease.
- a mutant DNA strand is synthesized and amplified by incubating the modified DNA strand in the presence of synthetic enzymes and oligonucleotides and a first degenerate oligonucleotide.
- the first degenerate oligonucleotide contains a codon for a different amino acid in the targeted site and in place of the amino acid in the active site in the known protease.
- the mutant DNA strand is then selected and screened by detecting the presence of the customized protease.
- the invention also includes a method of using customized proteases to modify a preselected substrate by transacylation.
- the transacylation reaction
- a customized protease ins incubated with a preselected substrate and a preselected nucleophile to form a mixture. The mixture is incubated sufficiently to form a preselected transacylation product, preferably in high yield.
- Customized proteases according to the invention can be utilized for post translation
- transacylation products produced are modified by
- nucleophiles including L-amino acids, D-amino acids, amino acid amides, amino acid derivatives, amino acid esters and radioactive amino acids or peptide derivatives including two or more amino acids of which the terminal amino acid is a natural amino acid or an amino acid derivative. It is understood that peptides produced by means other than recombinant technology can be transacylated according to the method of the
- mutant enzymes methods of making the customized proteases, as well as methods of using the customized proteases.
- the customized proteases of the invention are derived from known proteases and have transacylation capabilities differing from the known proteases.
- Altered transacylation capabilities include the
- mutant customized proteases have been modified so that the protease can perform transacylation reactions with different yields (i.e., 80 to 100%) or both.
- mutant enzymes can also be specifically designed and selected to perform transacylation reactions with a specific preselected substrate and/or nucleophile.
- a known protease preferably an exopeptidase, can be customized by replacement of amino acids in the active site so that the customized enzyme can transacylate different
- customized protease can also exhibit enhanced or
- substrates are preferably those that have an acidic or basic penultimate amino acid.
- Preselected nucleophiles are preferably acidic or basic amino acid amides.
- amino acids of the preselected substrates are designated by the letter "P".
- amino acids of the substrate on the N-terminal side of the peptide bond to be cleaved by a protease enzyme are designated
- Amino acids of the preselected substrate on the carboxy side of the bond to be cleaved by the protease are designated P 1 ', P 2 ', P 3 '...P n ' with P n ' being the amino acid furthest from the catalytic apparatus of the protease.
- the bond which is to be cleaved by the protease is the P 1 -P 1 ' bond.
- the generic formula for the amino acids of the preselected substrate are as follows: carboxypeptidase P n - - - P 3 - P 2 - P 1 - P 1 '
- the "active site" of the protease is divided into a number of substrate binding sites and a catalytic apparatus.
- the catalytic apparatus of serine proteases such as carboxypeptidase Y has a conserved catalytic triad of amino acids including serine, histidine and aspartic acid.
- the binding sites of the enzymes can include the S 1 binding site, the S 1 ' binding site which includes the C binding site.
- the S 1 binding site binds the side chain of the penultimate amino acid of the preselected substrate (P 1 ), the S 1 ' binding site binds to the side chain of the carboxy terminal amino acid (P 1 '), and the C binding site binds the terminal ⁇ r-carboxylate group.
- Modification of the active site preferably includes changes to amino acids in one or more of the binding sites.
- the terminology for the substrate binding site of a protease is analogous to that for describing the amino acids of the preselected substrate except that the substrate binding sites of the protease are designated by the letter "S".
- the substrate binding sites for the amino acids on the N-terminal side of the cleaved bond are labelled as S n ...S 3 , S 2 , S 1 .
- the substrate binding sites for amino acids on the carboxy side of the cleaved bond are designated by "S'”. These are labelled as S 1 ' S 2 ' ...S n '.
- the catalytic apparatus of the protease is understood to exist between the S 1 and S 1 ' substrate binding sites.
- a generic formula for describing substrate binding sites of a protease is:
- transacylation means that the enzyme can catalyze a reaction in which a leaving group is exchanged for a nucleophile.
- Transacylation reactions include
- Transpeptidation occurs when single or multiple amino acids or amino acid derivatives act as a leaving group and the nucleophile is a single amino acid or peptide or amino acid derivative.
- Peptide elongation involves replacement of a single amino acid with a peptide.
- Transacylation reactions also include peptide elongation if the leaving group is an alcohol and the nucleophile is a single or multiple amino acid unit.
- Customized proteases according to the invention can be utilized for post translation
- transacylation products produced are modified by
- nucleophiles including L-amino acids, D-amino acids, amino acid amides, amino acid derivatives, amino acid esters and radioactive amino acids or peptide derivatives including two or more amino acids of which the terminal amino acid is a natural amino acid or an amino acid derivative. It is understood that peptides produced by means other than recombinant technology can be transacylated according to the method of the
- the method of the invention provides for production of customized proteases through the process of site specific and/or random site mutagenesis.
- the invention further provides for selection and screening of suitably modified customized protease that is capable of catalyzing the preselected transacylation reaction.
- the method of the invention provides a means of producing customized protease through the process of traditional mutagenesis.
- the invention also provides a process for utilizing the customized protease to transacylate a preselected substrate to form modified peptide products.
- the invention provides for customized proteases.
- the customized proteases are derived from known proteases and have altered transacylating
- a customized protease is a modified version of a known protease designed to provide a protease that is capable of transacylating a preselected substrate with a preselected nucleophile in a transacylation reaction not substantially catalyzed by the known protease (i.e., less than 10%).
- customized protease can also exhibit improved or
- Improved or enhanced yields are yields preferably increased to about 40 to 80%, preferably about 80 to 100% over the yields of the known enzyme.
- Known proteases preferably include serine proteases, cysteine proteases and other endo- and exopeptidases. The especially preferred proteases are serine carboxypeptidases.
- Protease enzymes are capable of hydrolyzing preselected substrates as well as
- transacylating substrates in which the scissile bond is an ester and/or peptide bond is an ester and/or peptide bond.
- Transacylation can occur at the N or C-terminal end of the preselected substrate. While not meant to be a limitation of the invention, it is believed that an enzyme that catalyzes transacylation preferably can bind to or otherwise accommodate the nucleophile.
- the desired product of the reaction is designated the transacylation product.
- Yields of a desired transacylation product can depend on the (1) leaving group of the substrate, (2) the
- the competing side reactions can include (1) hydrolysis of the substrate, (2) hydrolysis of the desired
- undesirable side products through transacylation e.g., addition of nucleophiles to peptides originating from hydrolysis of the substrate, addition of nucleophiles to the desired transacylation product, polymerization of transacylation products, etc.
- Undesirable side products can include the hydrolysis product of the substrate, the hydrolysis product of the desired transacylation
- the preferred customized protease of the invention can catalyze the desired transacylation reaction in high yields (i.e., preferably about 40 to 100% and more preferably 80 to 100%), does not form substantial amounts of undesirable side products, and has a high affinity for the nucleophile.
- the preferred customized protease also does not substantially form undesirable transacylation side products, especially the
- transacylation product of a reaction of the hydrolysis product of the substrate with the substrate is substantially or that the undesirable side products preferably are about 0 to 40% of the yield and more preferably 0 to 20% of the yield and most
- undesirable side products can be removed from the reaction mixture by modifying the side product with an antigenic capping agent and removing the capped products with an affinity column.
- Side products can be modified with antigenic capping agents in a manner similar to that described in U.S. Patent No. 5,049,656 issued
- a preselected substrate is preferably not substantially transacylated by the known protease.
- the term substantially as used herein, means the yield of the transacylation reaction with the known protease is preferably about 0 to 40% and more preferably about 0 to 10%.
- the preselected substrate can be a naturally occurring peptide, a recombinant peptide, a synthetic peptide or a peptide in which the C-terminal ⁇ -carboxyl group has been esterified or otherwise modified.
- the preselected substrate has a core peptide connected to a leaving group at a terminus of the core peptide. The portion of the preselected substrate from which the leaving group is removed and to which the nucleophile is added is the core.
- Suitable leaving groups are amino acids, small peptides, or alcohols.
- the preferred leaving groups are small apolar or hydrophillic amino acids as well as moieties linked to the peptide core by an ester bond.
- the suitability of the preselected substrate is dependent on the substrate specificity of the
- the suitability of the leaving group is dependent on (1) the desired modification of the preselected substrate; (2) the substrate specificity of the customized protease; and (3) the manner in which the leaving group binds to the customized protease.
- a suitable preselected substrate for transacylation using a customized protease is of the general formula:
- P represents the N-terminal or C-terminal core of the preselected substrate and A is the leaving group.
- the leaving group (A) can be an amino acid, an amino acid amide, a peptide, a peptide amide, or an alcohol. If A is an amino acid, amino acid amide, peptide or peptide amide, cleavage of A, from the core (P) is at the peptide bond. If A is an alcohol, cleavage of A from the core is at the ester bond.
- the preselected substrate is not a substrate that is
- the preselected substrate has an acidic or basic penultimate amino acid.
- Suitable preselected substrates include growth hormone releasing factor (GRF) and derivatives thereof, calcitonin and derivatives thereof, and
- GLP-1 glucagon-like peptide-1 (SEQ ID NO:1).
- a nucleophile is a molecule that donates a pair of electrons to an atomic nucleus to form a covalent bond.
- a suitable nucleophile can be an amino acid derivative, peptide derivative, ammonia or labelled compound which can be added to the core of the preselected substrate by the customized protease capable of substituting the leaving group for the nucleophile.
- a suitable nucleophile can also include agents that can be converted to achieve the desired modification of the transacylation product. For example, photonucleophiles such as those described by Buckardt can be added to the substrate by transacylation with a customized protease and the resulting transacylation product can
- a suitable nucleophile can be preselected based upon (1) the desired modification of the final product; and (2) the ability of the nucleophile to displace the leaving group on the preselected substrate.
- nucleophiles include amino acids and amino acid derivatives such as amino acid esters and amino acid amides.
- Customized proteases are rendered suitable for a chosen transacylation reaction through modification of the known protease at the "active site.”
- Modifications of the mutant or customized enzyme can be site specific mutations designed to alter the "active site" of the protease so that it can act upon different preselected substrates and/or nucleophiles than the known protease. Modifications can include substitution, deletion, or insertion of one or more amino acids. The modifications can also be generated by random mutagenesis.
- amino acids in the active sites of proteases are known to those of skill in the art.
- amino acids equivalent to amino acids in known binding sites of proteases can be identified using standard methods. These methods include identification of equivalent amino acids by reference to the primary and/or tertiary structure of an enzyme in that class of proteases. For example, a reference enzyme for
- carboxypeptidases is wheat carboxypeptidase (CPD-WII). The primary amino acid sequence and the crystal
- CPD-WII structure of CPD-WII are known (Liao and Remington, J. Biol. Chem., 265:6528 (1990)) and can serve as reference points to identify equivalent amino acids in other carboxypeptidases.
- CPD-Y The amino acid sequence and crystallographic structure of CPD-Y are known as well (Endrizzi et al., Biochemistry, 33:11106 (1994)) and can similarly be used as reference points to identify equivalent amino acids in other carboxypeptidases.
- One method that is applied to identify residues in the active site of a protease with an unknown tertiary structure is comparison of the amino acid sequence of the protease of interest with the amino acid sequence of a homologous protease with a known tertiary structure.
- this method can be used to identify amino acids in the protease of interest that are equivalent to amino acids in the active site of the homologous protease. For example, see Olesen et al.
- amino acids in the active site can be identified by determination of the tertiary structure using X-ray crystallography or NMR techniques.
- carboxypeptidase Y is modified by substitution of amino acids in the active site. These amino acids are preferably found in the S 1 or S 1 ' binding sites. Preferred amino acids of the S 1 binding site include Tyr147, Leu178, Tyr185, Tyr188, Asn241, Leu245, Trp312, Ile340 and Cys341. Preferred amino acids of the S 1 ' binding site include Trp49, Asn51, Gly52, Cys56, Thr60, Phe64, Glu65, Glu145, Tyr256,
- Amino acid substitutions in the S 1 binding site can preferably result in a mutant protease capable of transacylating a preselected substrate with a basic or acidic penultimate amino acid (P 1 ).
- substitutions in the S 1 ' binding site can preferably result in a mutant protease capable of performing transacylation reaction on preselected substrates with large apolar amino acid leaving groups and/or
- nucleophiles such as large apolar amino acids, proline and proline amide.
- the especially preferred enzyme is a customized carboxypeptidase that has different amino acid residues in a position equivalent to amino acid residue 178 or 398 of carboxypeptidase Y.
- a preferred substituent amino acid is serine at position 178.
- the preferred customized protease is a carboxypeptidase that is capable of transacylating a preselected substrate having an acidic or basic amino acid such as growth hormone releasing factor with a
- GRF C-terminal alanine (GRF (1-43)-Ala) (SEQ ID NO:2) and arginine as the penultimate amino acid.
- the especially preferred mutant carboxypeptidase catalyzes formation of growth hormone releasing factor with a leucine amide.
- Selection can also involve choosing the different amino acid that will be substituted into the active site. While not meant to be a limitation of the invention, one way the amino acid to be inserted into the active site can be selected is by predicting the effect on the binding interaction between the
- substitutions can be conservative amino acid substitutions.
- preselected substrate and/or nucleophile can directly affect the transacylation process.
- enzyme substrate effect formation of the enzyme substrate complex (ES) which is a first step to transacylation.
- enzyme substrate complex For serine or thiol proteases, enzyme substrate
- acyl- or thio-acyl species then undergoes nucleophilic attack (aminolysis) to form the transacylated product.
- the binding interaction involved in formation of the ES complex include three major types:
- transacylation capabilities of the protease through changes in the binding affinity of an enzyme for a substrate (i.e. formation of ES complex), through modification of the interaction of the enzyme with the transition state as well as through interactions
- Amino acid substitutions that affect each of these stages can be predicted based upon the preselected substrate with leaving group and the preselected nucleophile.
- the preferred substitutions include the substitution of Asn51 with glutamine and Leul78 with serine in carboxypeptidase Y.
- the mutant or customized protease can also be a protease that exhibits enhanced transacylating
- Enhanced transacylating capability can be determined by determining an increase in the yield of the transacylation product.
- the increase in the yield is about 40 to 100% and more preferably about 80 to 100% increase over the yield catalyzed by the known protease.
- the preselected substrate can either be a substrate that can be transacylated by the known protease but at low yields (i.e., about 10-40%) or a substrate not substantially cleaved by the known protease (i.e., less than 10%).
- the method of the invention provides for preparing a customized protease derived from a known protease and that has a modified active site and that functions to alter the transacylation capability of the known protease.
- the customized enzymes can be modified to transacylate a new substrate by mutating one of more amino acids in one or more of the substrate binding sites of a known protease. It is possible that mutation of as few as one amino acid in one substrate binding site can provide for
- transacylation of a substrate that was not a suitable substrate for the known protease The active site can also be modified to provide an enzyme having enhanced transacylation capability, i.e., higher yields of transacylation.
- Mutation of amino acids of the substrate binding sites can alter one or more functionalities which affect transacylation of a preselected substrate by a customized protease such as: (1) affinity of the customized protease for the core peptide portion of the substrate; (2) affinity of the customized protease for the leaving group or nucleophile; and (3) preference of catalysis of aminolysis over the competing hydrolysis reaction.
- mutation of a known protease to produce a customized protease can be accomplished through site specific mutagenesis, random site mutagenesis and traditional mutagenesis.
- the first two methods require knowledge of the DNA sequence of known proteases and the location of codons which code for the substrate binding site amino acids.
- Amino acids in the active site of proteases are either known to those of skill in the art or can be identified by analogy to known proteases as described herein.
- the corresponding DNA sequence encoding the known protease and the location of codons for amino acids in the active sites are either known to those of skill in the art or can be derived from the amino acid sequence. For example, the DNA sequence and
- DNA sequences encoding known proteases can be obtained from an electronic database such as SwissProt, GeneBank, and EMBL. Once these sequences are
- publications identifying vectors containing the DNA sequence can be located and used by those of skill in the art to prepare a customized protease.
- the putative DNA sequence can be derived from the amino acid sequence of the known protease.
- the amino acid sequence of the known protease can be used to prepare synthetic oligonucleotide probes.
- the probes can be used to identify DNA sequences encoding the known protease in suitable organisms by standard methods as described by Maniatis et al, A Guide to Molecular Cloning (1989). Once a DNA sequence encoding a known protease is isolated, codons corresponding to amino acids in the active site can be identified as described herein.
- Active site amino acids can be identified by comparing the primary or tertiary structure to other known active sites of other proteases or by X-ray crystallography as described herein. b. Modifying amino acids in the active site
- Amino acids in the active site can be modified by modifying the codon encoding the amino acids in the DNA sequence encoding the known protease. Amino acids in the active site and the location of codons encoding these amino acids are either known to those of skill in the art or can be determined using standard methods. Amino acids in the active site preferably include those found in the S 1 , S 1 ' or C binding sites. The codon or codons encoding amino acids of the active site of the protease are included in a targeted site on the DNA sequence. The targeted site includes the DNA sequence that is going to be mutated. One or more than one codon can be changed in the targeted site. Optionally, the targeted site can also include the DNA sequence
- the DNA sequence of the targeted site surrounding the codon preferably includes about 3 to 9 nucleotides on either side of the codon or codons for amino acids in the active site. Modifications of codons include substitution, insertion or deletion of the codon.
- the codons encoding amino acids of the active sites are preferably modified to encode a different amino acid than that of the known protease.
- site specific modification a selected amino acid in the active site can be changed either randomly or by
- the preferred codons for modification are those that encode amino acids in the S 1 or S 1 ' binding sites of carboxypeptidases. While not meant to be a limitation of the invention, it is believed that
- preselected substrate with a basic or acidic penultimate amino acid (P 1 ).
- P 1 a modification of amino acids in the S 1 ' binding site can result in a mutant protease capable of transacylating a preselected substrate with an acidic or basic leaving group and/or amino acid amides as nucleophiles.
- Choice of a specific amino acid substitution at a random or specific location can be based on the known or inferred mechanism of interaction of the binding site amino acid and the substrate. From this a rational inference is made, using knowledge of the properties of amino acids, of what amino acid
- substitution will provide the appropriate interaction to effect transacylation of the preselected substrate.
- amino acid properties which may be considered when selecting specific amino acid substitutions include electronic and steric factors.
- specific amino acid substitution selection may be based on pKa values (of ⁇ -carboxyl and side chain hydrogens), amino acid side chain length, and amino acid side chain polarity at various pH. While not meant to be a
- the effect of the amino acid substitution can be predicted based upon the interactions involved in binding and catalysis as described herein for carboxypeptidase.
- random selection of amino acid substitutions can be made both with respect to the amino acids of the active site to be changed and the amino acid substitutions to be made.
- site specific and random site mutagenesis are used to mutate the known protease and can be accomplished through incorporation of an oligonucleotide containing a mutated or modified codon at the chosen or targeted codon location.
- Other methods of random and site specific mutagenesis can be employed as described by Maniatis, cited supra.
- Preferred methods of incorporation of the oligonucleotide into the DNA sequence encoding the known protease to produce a modified DNA sequence include polymerase chain reaction (PCR) and standard cloning technology.
- Oligonucleotides containing a mutated or modified codon can be obtained by standard methods.
- oligonucleotides are comprised of a variable and a constant region and preferably are about 20 to 60 nucleotides long.
- the length of the oligonucleotide is dependent on two main factors; (1) the number of variable regions the oligonucleotide is coding for; and (2) the length of the constant regions.
- variable region of the oligonucleotide contains the nucleic acid codons which code for the mutated amino acids of the substrate binding sites.
- the codons for amino acids are known to those of skill in the art.
- the variable region of oligonucleotide can be designed to include a codon for a specific amino acid or any number of random amino acids. Therefore, the minimum number of codons in the variable region is three, which represents the codon for a single amino acid.
- the codons of the variable region correspond to the location of the codons to be mutated in the known protease.
- the variable region is flanked by the
- oligonucleotide contains more than one variable region, there are constant regions between variable regions.
- the constant regions are necessary to incorporate the oligonucleotide into the customized protease gene and include codons corresponding to those of the known protease at that location (i.e., that are not mutated).
- the length of the constant region can depend on the means by which the oligonucleotide will be incorporated into the customized gene and the number of amino acid modifications included in the variable region.
- the constant region includes about 3 to 50 nucleotides on either side of the variable region, and more preferably about 3 to 30 nucleotides on either side of the variable regions.
- the synthetic oligonucleotides are incorporated into the DNA sequence for the known protease in frame and at the targeted location.
- This insertion can occur is by cleavage with at least one appropriate restriction endonuclease so that the targeted site is deleted, followed by ligation of the synthetic oligonucleotide into the site that was
- restriction endonucleases can be determined by examining the nucleotide sequence around the targeted site and by the size of the synthetic oligonucleotide to be inserted at the site.
- recognition sequences of restriction enzymes are known to those of skill in the art, and an appropriate
- the codon for Asn51 of carboxypeptidase Y is modified to encode glutamine 51.
- the PCR1 gene encodes carboxypeptidase Y and can be obtained from plasmid pTSY3 which has been deposited with the American Type Culture Collection in Rockville, MD on October 26, 1993 and given Accession No. 75580.
- An oligonucleotide including a codon for glutamine at a site corresponding to the codon for Asn51 such as:
- the BamHI fragment of PRC1 includes the codon for amino acid 51.
- the DNA sequence of carboxypeptidase Y surrounding the codon for Asn51 can be deleted from the BamHI fragment of PRC1 with restriction endonucleases such as BstXI and SmaI.
- the synthetic oligonucleotide can then be introduced in place of the deleted DNA sequence at the SmaI-BstXI site of the BamHI fragment.
- the modified BamHI fragment is then inserted back into the entire DNA coding sequence for carboxypeptidase Y using known methods to form a modified DNA sequence.
- the sequence of the modified DNA sequence can be confirmed using dideoxy sequencing methodology.
- the modified DNA sequence can be introduced into a suitable host cell, selected and expressed to yield the customized protease with the modified active site and that functions to alter the transacylation activity of the known protease.
- the modified DNA sequence is preferably incorporated into a vector to provide for selection and expression.
- Suitable vectors include the yeast
- bacterial shuttle vectors YEp24, pRA21 ⁇ BAM, pYSP1, pTSY3, pRA21, and pYSP32.
- the modified or mutated DNA sequence can be incorporated into the vectors by
- Suitable host sells include bacteria such as E. Coli and yeast such as S. cerevisiae.
- Preferred host cells include S. cerevisiae strains having isogenic vp1 mutations, delta-prc1 mutations and ura3 mutations.
- Especially preferred hosts are S. cerevisiae strains that have vpl mutations resulting secretion of active CPD-Y as described in Nielsen et al., cited supra.
- the preferred vector is a plasmid pTSY3 which is the yeast bacterial shuttle vector YEp24 with a
- Suitable host cells are transformed by
- Transformed cells can be selected- based upon the
- Transformed yeast cells can be screened for the production of mutant protease activity.
- Transformed yeast cells producing mutant proteases can be screened by detecting the ability of the transformed cells to hydrolyze a peptide substrate using standard methods as described by Nielsen et al., cited supra.
- nucleophile can be further selected by assaying for transacylation activity by standard methods including those described in Examples 2 and 3.
- mutant proteases can be purified using standard methods such as high performance liquid
- a novel method of the invention involves mutagenizing a known protease to form a customized protease having altered transacylating capability.
- the basic technique of the method involves in vitro DNA synthesis primed by mutagenic degenerate synthetic oligonucleotides. The method provides
- the steps of the method include providing a DNA sequence encoding a known protease.
- D ⁇ A sequences encoding known proteases are either publicly available or can be obtained by standard methods as described herein.
- a targeted site of the D ⁇ A preferably
- the D ⁇ A sequence includes at least one codon for an amino acid of the active site to be modified as described herein. Once targeted sites are identified, the D ⁇ A sequence is modified at each targeted site by insertion of stop codons and optionally restriction endonuclease sites at the location to be mutated. Codons for stop codons are designated amber, ochre and opal, and the sequences of the stop codons are known to those of skill in the art. The D ⁇ A sequences recognized by a restriction
- the D ⁇ A sequence including the restriction endonuclease site can be adjacent to the stop codon or it can overlap with the stop codon.
- the oligonucleotide sequence inserted at the target site can be prepared by standard methods including automated D ⁇ A synthesis.
- the inserted oligonucleotide sequence is preferably about 3 to 60 nucleotides long and can be inserted into one or more targeted site using standard methods such as in vitro D ⁇ A synthesis as described by Maniatis et al, cited supra.
- endonuclease site is inserted into a targeted site of the D ⁇ A encoding the known protease, a modified D ⁇ A sequence encoding an inactive known protease is formed.
- the presence of stop codons results in the expression of truncated forms of the known protease lacking activity.
- the D ⁇ A sequence encoding an inactive known protease is introduced into a vector, preferably a phagemid vector.
- the vector is transformed into suitable host cells such as E. coli for amplification. Once amplified, the vector is isolated and single stranded DNA can be prepared.
- the DNA sequence can be introduced into a vector carrying an inactive antibiotic resistance gene such as a gene encoding ampicillin resistance that has a frameshift mutation.
- a preferred phagemid vector is the pYSP1.
- a mutant DNA strand encoding the customized protease can be synthesized by incubating the single stranded DNA with one or more first degenerate
- a first degenerate oligonucleotide has variable and constant regions as described previously herein.
- oligonucleotide includes at least one mutated codon for an amino acid in the active site of the known protease and that has been targeted.
- the mutated codon is found at the same location with respect to the surrounding DNA as the codon for the amino acid in the known protease.
- An oligonucleotide is degenerate if the mutated codons are randomly changed to encode any one of the 20 amino acids.
- a degenerate oligonucleotide at the mutated codon has the sequence of NNN wherein N corresponds to any one of the four nucleotides.
- oligonucleotide preferably includes about 10 to 50 nucleotides on both sides of the mutated codon.
- the codons of the constant region correspond to the codons of the known protease at the targeted location in the known protease.
- Degenerate oligonucleotides can be for any of the 20 amino acids and are randomly generated using known methods and as described in Olesen et al., cited supra.
- DNA sequence encoding the known protease contains more than one targeted site that has been modified by stop codons and/or restriction
- the other targeted sites in the mutant enzyme should have the sequence of the known enzyme at these other targeted sites.
- One or more second oligonucleotides can be included in the DNA synthesis mixture that function to ensure that the other targeted sites that are not to be mutated in the mutant DNA strand have the sequence of the known enzyme as described in Olesen et al., cited supra .
- the second oligonucleotides include codons corresponding to those in the known protease that have been replaced by stop codons and optionally restriction endonuclease sites at targeted sites. Each second oligonucleotide has the same sequence at a specific targeted site as the known protease.
- oligonucleotide preferably has about 20 to 60
- the synthesis mixture can also optionally include an oligonucleotide that provides for repair of an antibiotic resistance gene.
- an oligonucleotide that provides for repair of an antibiotic resistance gene.
- oligonucleotides that can repair the mutation of the antibiotic resistance gene in the DNA synthesis mixture results in a mutant DNA strand that has a functional antibiotic resistance gene.
- resistance gene includes codons that provide for the correct DNA sequence at the relevant mutation in the resistance gene.
- These oligonucleotides are known to those of skill in the art or can be prepared by standard methods as described in herein.
- mutant DNA Once synthesis of the mutant DNA is complete, desirable mutant enzymes encoded by the DNA can be selected and screened for the ability to act on a preselected substrate for alteration of transacylation capability.
- the mutant DNA can be selected and
- a suitable host cell such as E. coli followed by introduction into a suitable host cell that can secrete proteases such as S. cerevisiae strains.
- Selection methods for transformed cells include
- selecting for antibiotic resistance based upon the presence of an antibiotic resistance gene on a vector.
- Methods for selection in yeast using selectable marker genes are known to those of skill in the art.
- Transformed cells can be screened to identify cells having the customized protease with the desired functional activity.
- the desired customized protease can include a protease that can hydrolyze a preselected substrate having an acidic or basic penultimate amino acid, a protease that can catalyze transacylation reaction in which a preselected substrate is modified by a preselected nucleophile, and a protease with enhanced transacylation capabilities.
- the preferred customized protease is a carboxypeptidase that can modify a
- preselected substrate with a basic penultimate amino acid with a terminal leucine amide.
- hydrolyzing a preselected substrate are first selected and screened for hydrolysis activity and then those selected mutants are screened for transacylation
- Substrate hydrolysis is used as a first level selection to ensure that the mutant is capable of acting on the preselected substrate.
- the mutant enzymes capable of hydrolyzing the preselected substrate are then further screened for transacylating capability. Selection of transformants expressing hydrolysis
- activity of the preselected substrate can be performed using the plate activity method or the color overlay method.
- the plate activity method or the color overlay method For detection of low level customized
- transformed cells expressing customized proteases can be selected for preselected substrate hydrolysis activity using a plate activity method.
- This method of the invention utilizes a bacterial or yeast host cell which requires an amino acid for growth. The amino acid required for growth is provided to the transformed host cells as a C-terminal amino acid of a peptide. By culturing the transformants on a media deficient in the leaving group amino acid, only those transformants capable of releasing the leaving group from the peptide substrate can grow. This method is described in Olesen et al., Protein Eng., cited supra. For example, a vps strain of S.
- a preferred method for selection of transformants which express a customized protease capable of hydrolyzing a preselected substrate is the color overlay method.
- transformant colonies are overlaid by agar containing a chromogenic substrate which reveals customized protease activity.
- the chromogenic substrate turns color upon reaction with the product formed by catalytic action of the customized protease.
- the transformed cells are incubated with a preselected peptide or amino acid substrate.
- the preselected peptide or substrate such as N-acetyl-L-alanine
- ⁇ -naphthyl ester is acted upon by transformed cells expressing a customized protease and the reaction product is detected by simultaneously overlaying the transformed cells with a chromogenic agent such as Garnet Red that changes colors upon exposure to the reaction products.
- a chromogenic agent such as Garnet Red that changes colors upon exposure to the reaction products.
- Customized proteases which have been mutated and screened for the capacity to act on a preselected substrate can be further screened for transacylation capability.
- One method that can be employed to screen for transacylation capability is that described in Examples 2 and 3.
- Customized proteases are purified from the transformed cells by methods known in the art and as described in Examples 2 and 3. The purified customized protease is mixed with the preselected substrate under conditions favorable for transacylation such as pH of about 7 to 9.5 and in the presence of a suitable nucleophile.
- transacylation of the preselected substrate can be identified by following the appearance and/or amount of the desired reaction product by standard methods.
- the PRC1 gene encoding carboxypeptidase Y is modified by insertion of DNA sequences at target sites as follows:
- TAG CCC GGG TGT SEQ ID NO:7 at Glu214, Arg216
- the target sites in carboxypeptidase were selected based on homology to carboxypeptidase-WII.
- the modified DNA sequence encodes an inactive carboxypeptidase Y.
- DNA sequence as modified is amplified as phagemid vector pYSP32 in E. coli.
- the preferred vector also contains an inactive frame shifted ampicillin resistance gene.
- Single stranded DNA of pYSP32 can be generated by standard methods.
- the single stranded DNA can be incubated with up to four first degenerate oligonucleotides, each 20 to 60 nucleotides long and containing degenerated codons at the center.
- a preferred first degenerate oligonucleotides each 20 to 60 nucleotides long and containing degenerated codons at the center.
- oligonucleotide for position 178 includes the sequence:
- NNN is situated at the center of the
- oligonucleotide and corresponds to the location of the codon for amino acid 178.
- NNN is a codon for any of the 20 amino acids.
- Degenerate oligonucleotides can be synthesized by automated synthesis.
- One or more second oligonucleotides can be included in the preferred synthetic mixture and in which case they each replace one of the first
- the second oligonucleotide includes codons for amino acids at positions 147, 178, 215 and 216, and 340 and 341 of carboxypeptidase Y, as follows:
- TTC ATC TGT ACC (SEQ ID NO:11) Ile340, Cys341
- One preferred mutagenesis mixture contains a first degenerate oligonucleotide for position 178 and a second oligonucleotide for each position of Tyr147, Glu215, Arg216, Ile340 and Cys341.
- the preferred DNA synthesis mixture also contains one or more third
- the repair oligonucleotides provide for synthesis of a mutant DNA sequence having a functional ampicillin resistance gene operably linked to the DNA sequence encoding the mutant or customized protease.
- the mixture of single stranded DNA and oligonucleotides is incubated in the presence of
- Mutant DNA sequences encoding the customized protease are formed and can optionally be linked to a functional ampicillin resistance gene.
- the mutant DNA is
- the preferred suitable yeast cell is a S. cerevisiae strain that has a vps mutation, and/or requires at least one amino acid for growth. Especially preferred S.
- cerevisiae strains include W2579, K2579LLR and JHRY20-2C ⁇ 3.
- the transformed E. coli cells can be selected first for the ability to grow in the presence of
- transformants are further selected for the ability to grow on a medium deficient in the amino acid required for growth and in the
- a host cell that cannot normally grow without leucine is transformed with mutant DNA and plated onto leucine deficient medium supplement with a preselected peptide having the following formula:
- protease such as any one of Glu, Gly, Ser, His, Pro, Trp or Lys.
- Transformants that can grow on leucine deficient medium supplement with a preselected polypeptide such as P n -Lys-Leu can act on the preselected polypeptide to release leucine, thereby providing a source of leucine for growth.
- These expressed proteases are then screened for the ability to transacylate a preselected polypeptide such as P n -Arg-Ala where P n is growth hormone releasing factor and leucine amide.
- Customized proteases which favor aminolysis over hydrolysis can be produced using traditional mutagenesis.
- mutation of the amino acid composition of a known protease is accomplished by subjecting DNA or cells containing a vector encoding a DNA sequence for known protease to a mutagenic agent such as UV light,
- nitrosoguanidine ethylmethyl sulfonate, bisulfite, dimethyl sulfate, formic acid, hydrazine, hydroxylamine, methoxylamine, nitrous acid, potassium sulfate, and others.
- Methods of traditional mutagenesis are known in the art and are described, for example: for chemical in vi tro mutagenesis : Myers et al., Science, 229:242-247 (1985); Hayatsu, Methods Enzymol., 45:568-587 (1976); Shortle et al., Methods Enzymol., 100:457-568 (1983); Kadonga et al., Nucl. Acids. Res., 13:1733-1745 (1985); Busby et al., J. Mol. Biol.. 154:197-209 (1982); and Loeb, Cell, 40:483-484 (1985); for nucleotide
- the mutated vectors are then incorporated into a suitable expression system and the expressed
- customized enzymes are selected and screened.
- the methods for selecting and screening customized protease produced by traditional mutagenesis can be performed as described above for transformants produced by site specific and random site mutagenesis. Plate activity and color overlay selection can be utilized to select for those transformants which express a customized protease capable of acting on a preselected substrate.
- Those customized proteases capable of acting on a preselected substrate are purified and mixed with a preselected substrate and nucleophile under conditions favorable for transacylation to screen for enzymes capable of catalyzing the transacylation of the
- the invention also provides a method for using the customized enzyme to transacylate a preselected substrate with a preselected nucleophile.
- This method is useful to add nucleophiles such as D-amino acids, modified amino acids and radiolabelled amino acids to the termini of recombinantly produced peptides to form transacylation products.
- This method can also be applied to naturally occurring or synthetic peptides to form useful analogs or derivatives.
- the customized protease of the invention is designed to either enhance transacylation capabilities (i.e., yields) or act on a preselected substrate and/or nucleophile poorly accepted by the known proteases.
- the customized protease can be prepared and selected by the methods described herein.
- the preselected substrate is selected depending on the desired transacylation
- the preselected substrate is preferably not substantially transacylated by the known proteases.
- substantially in this context means that the yields of transacylation with the preselected substrate and with a particular nucleophile are preferably about 0 to 10% under standard conditions.
- the preselected substrate preferably has a basic or acidic amino acid as the penultimate amino acid. The preferred amount of the preselected substrate depends on the substrate
- a preselected substrate can be a naturally occurring peptide, a synthetic peptide or a recombinantly produced peptide.
- the preselected nucleophile is preferably not an effective nucleophile with the known protease.
- the nucleophiles are preferably amino acids, radioactively labelled amino acids, and amino acid amides.
- Nucleophiles can be prepared by standard synthetic methods known to those of skill in the art such as described in Breddam et al., Int. J. Peptide Res.,
- a preferred amount of a nucleophile also depends on the affinity of the enzyme and
- solubility of the nucleophile in the chosen solvent is about 10 mM to 2M.
- Reaction conditions resulting in high yields of the desired product can vary with a given enzyme substrate system. Reaction conditions can be altered to minimize degradation and polymerization of the products. Such side reactions may, when using ester substrates together with a serine carboxypeptidase, be avoided by increasing the pH above 8.0 when aqueous solvents are employed. Alternatively, side reactions can be avoided by conducting the reaction in an organic solvent.
- Transacylation can be performed in aqueous buffer solution.
- Preferred buffer solutions include 50mM HEPES and 5mM EDTA, pH 7.5 or 50mM CHES and 5mm EDTA, pH 9.5. It is of importance that the chosen buffer is unable to act as a nucleophile in the
- the preferred pH for transacylation using an amino acid or peptide derivative leaving group is preferably about pH 5.5 to 8.5, and more preferably about preferably pH 6.5 to 7.5.
- the production of the transacylation product is monitored by HPLC or other appropriate analytical technique.
- the reaction can be stopped by addition of an acidic solution to bring the pH of the reaction mixture down to about pH 1 to 3.
- the reaction can be stopped by addition of an enzyme
- the transacylation product can be separated from the
- reaction mixture by reverse phase chromatography, hydrophobic interaction chromatography, ion exchange chromatography, or HPLC.
- the transacylation reaction can be performed in organic solvents for those enzymes substrate systems capable of functioning in organic solvents.
- transacylation reaction include dimethyl sulfoxide
- the preselected peptide substrate GRF (1-43)-Ala (SEQ ID NO:2)
- GRF (1-43)-Ala SEQ ID NO:2
- the nucleophile, leucine amide is dissolved in 50 mM HEPES, 5 mM EDTA to a final concentration of 500 mM.
- 25 ⁇ l of a 40 mM solution of GRF (1-43)-Ala (SEQ ID NO:2) is added pr. 950 ⁇ l of nucleophile solution and the pH is add to 7.5 at 20°C.
- the customized protease is added to the mixture in 25 ⁇ l of water pr. ml
- carboxypeptidase Y suggests that the side chains of Trp49, Asn51, Gly52, Cys56, Thr60, Phe64, Glu65, Glu145, Tyr256, Tyr269, Leu272, Ser297, Cys298 and Met398 are important in the active site of the enzyme. These amino acid residues were mutated by site specific mutagenesis to form enzymes with single, double, or triple
- N51E Glutamic Acid
- N51Q Glutamine
- N51S Serine
- Glu 65 has been replaced by: Alanine (E65A)
- Glu145 has been replaced by: Alanine (E145A)
- mutant enzymes are by site specific mutagenesis using the polymerase chain reaction.
- a plasmid pUC- ⁇ 30 was constructed by inserting a 1112 bp BamHI fragment of the PRC1 gene from pYTS3 containing the coding region for all amino acid residues involved in the formation of the active site into the unique BamHI site in the polylinker of pUC19. Yanisch-Perron et al., Gene, 33:103 (1985);
- pUC- ⁇ 30 contains unique BstXI, EcoRI, NaeI and SmaI restriction sites which can be used in cloning and mutagenesis procedures.
- the mutations W49F and N51A were made by the polymerase chain reaction (PCR) (Innes et al., 1990) in a Perkin Elmer Cetus DNA Thermal Cycler using a Gene Amp kit (Perkin Elmer Cetus) on pUC- ⁇ 30 with
- GTTTCTGTCCTTGTGAGACAAAATTTCAGA (SEQ ID NO:13) (oligo wtl1) and with either GGATCCGGTCATCCTTTTCTTGAACGGG (SEQ ID NO: 14) (oligo W49F) or
- oligo N51A Nucleotides underlined are different from wild-type. Cleavage with BstXI allowed insertion of the PCR fragment into a SmaI-BstXI vector fragment of pUC- ⁇ 30.
- the mutation E145A was made by PCR with
- GCAAGGCGATTAAGTTGGGT (SEQ ID NO:16) (oligo pUC19 sp1) and GGCGTAGGAAGCCCCAGCGAT (SEQ ID NO:17) (oligo E145A) on pUC- ⁇ 30.
- Cleavage of the PCR fragment with EcoRI allowed introduction into a NaeI-EcoRI vector fragment of pUC- ⁇ 30.
- the mutations E65A and N51A+E65A were produced by fusion of two overlapping PCR fragments using either pUC- ⁇ 30 or pUC- ⁇ 30-N51A as template.
- Fragment 1 was generated with CTGTTCTTTGCATTAGGACCC (SEQ ID NO:18) (oligo E65A) and (oligo wt1) and fragment 2 with (oligo pUC19sq1) and (oligo E145A).
- An additional PCR reaction was performed on the fused fragment with oligo pUC19sq1 and oligo wt1.
- the resultant fragment was cut with EcoRI and BstXI, thus, removing the unwanted mutation on position 145, and ligated into a pUC- ⁇ 30 vector fragment cut with the same restriction enzymes.
- N51A+E145A, E65A+E145A, and N51A+E65A+E145A were made by proper combination of the above listed mutations exploiting the EcoRI site in the polylinker and exploiting that BstXI cleaves between position 65 and 145.
- the mutated sequences were introduced into the PRC1 gene by transferring the mutated 1112 bp BamHI fragment into the vector pRA21 ⁇ Bam.
- the fragment inserted into pRA21 ⁇ Bam was controlled for the absence of any non-silent secondary mutation by sequencing using the Taq Dye-DideoxyTM terminator cycle sequencing kit and the model 373A DNA-sequencing system from Applied
- Site directed mutagenesis on position 51 and 145 was performed using polymerase chain reaction and restriction endonuclease cleavage as described herein. The following oligonucleotides were used in the
- GGCGTAGGATGACCCAGCGAT (SEQ ID NO:27) (oligo E145S). Underlined nucleotides are different from wild-type. All fragments generated by the PCR reaction were ligated into pUC- ⁇ 30 after cleavage with the appropriate
- restriction enzymes such as EcoRI (E145) or BstXI
- mutant enzymes containing cysteine (N51C), valine (N51V), or glycine (N51G), glutamine (E65Q), and asparagine (E145N) were prepared in a similar manner.
- Mutant enzymes were purified from a one liter culture grown under the conditions previously described. (Nielsen et al., 1990). Growth media containing
- the elute was concentrated -using an Amicon cell and applied to a Sephacryl-S300 column (1 cm ⁇ 100 cm) equilibrated with 50 mM NaH 2 PO 4 , pH 7.0. Fractions with constant specific activity were pooled, concentrated and dialyzed against water. All enzyme preparations were stored frozen in water at -18°C.
- the purity of the mutant enzymes was ascertained by SDS-PAGE on 12.5% homogeneous gels using the PhastSystem from Pharmacia.
- the mutant or customized enzymes can be evaluated for a change in the
- carboxypeptidase Y (CPD-Y) for the negatively charged C-terminal carboxylate group of peptide substrates has been identified using site directed mutagenesis as described herein. While not meant to be a limitation of the invention, it is
- the carboxylate group of the peptide substrate binds to the side chains of Asn51 and Glu145 in the S 1 ' binding pocket. Both side chains can act as hydrogen bond donors.
- the side chains of Asn51 and Glu145 appear to be oriented by hydrogen bonds with Glu65 and Trp49 which, therefore, have an indirect function in the binding of the carboxylate group of peptide substrates.
- Serine carboxypeptidases also catalyze the hydrolysis of peptide esters and this activity increases with pH and remains constant in the pH range 7 to 9.5. Thus, at basic pH, the esterase activity is high and the peptidase activity is low.
- mutant carboxypeptidase enzymes can bind to and catalyze peptide elongation with amino acids as nucleophiles in higher yields.
- yields exceeding 60% are obtained in a few cases but yields of 10-40% are much more common and H-Pro-OH, H-Glu-OH and H-Asp-OH are not accepted as nucleophiles.
- the yields obtained with amino acids as nucleophiles are rarely satisfactory.
- the low yields with amino acids as nucleophiles are not due to degradation of the product since the reaction is carried out at basic pH where the peptidase activity is very low (see above), thus, securing accumulation of the peptide product in the reaction mixture.
- Mutants of carboxypeptidase Y were examined for the capacity to transacylate certain substrates using amino acids as nucleophiles. Some amino acid substitutions in the active site of mutant
- carboxypeptidase Y enzymes were also made knowing that they were not likely to improve the yields of
- CPD-Y was obtained from Carlbiotech
- Example 1 The purity of the enzymes was ascertained by SDS polyacrylamide gel electrophoresis.
- the reactant composition was determined by HPLC using a Waters HPLC equipped with a C-18 Waters Novapac 4 ⁇ reverse phase column and various gradients of
- the composition of the reaction mixture was determined at least twice during the reaction, the first time when 20-50% (preferably 35%) of the ester substrate had been consumed in the reaction and the second time when 50-90% (preferably 80%) of the substrate had been consumed.
- the products were collected and identified by amino acid analysis after acid hydrolysis using a
- the fraction of aminolysis (fa) was expressed as the ratio between the formed aminolysis product and the sum of all products being formed, i.e., unc ⁇ nsumed substrate was disregarded in the calculations.
- the K N(app) representing the nucleophile concentration at which fa is half the maximum value (a measure for the dissociation constant of the nucleophile), and fa max (the highest possible fa obtained at saturation of the enzyme with nucleophile) were determined by fitting the values of fa obtained at a minimum of seven concentrations of nucleophile to a saturation. The value of fa obtained at the highest possible nucleophile concentration is designated fa sat .
- transacylation reactions with amino acids or amino acid derivatives acting as nucleophiles in competition with water can be studied.
- aminolysis is preferably performed with an ester
- Transacylation reactions should preferably be performed at slightly basic pH to maximize the esterase activity and minimize the peptidase activity.
- amino acids are used as nucleophiles the product peptide is very slowly degraded by the enzyme and, as a consequence, it accumulates in the reaction mixture,
- FA-Ala-OBzl is hydrolyzed at very high k cat /K M by CPD-Y and the prepared mutant enzymes allowing the use of low concentrations of enzyme (0.5 ⁇ g/ml).
- the fact that the peptide products are hydrolyzed at much lower k cat /K M prevents degradation of the aminolysis product.
- pH 7.5 with H-Val-OH as added nucleophile, two products were formed: FA-Ala-OH (hydrolysis) and FA-Ala-Val-OH (aminolysis).
- the fraction undergoing aminolysis reaction was constant with time and independent of the
- nucleophile (amino/ammonium) has little influence on the synthesis parameters.
- fa max consistently exceeds 0.85 and is essentially independent of the hydrophobicity of the side chain.
- ⁇ -carboxylate group of some amino acid nucleophiles apparently had an adverse effect on fa max suggesting that alteration of this interaction could have a beneficial effect.
- Amino acid nucleophiles could occupy a position similar to that of the P 1 ' amino acid residue of peptide substrates. If this is the case, then the amino acids in CPD-Y involved in the binding of the C-terminal carboxylate group of peptide substrates would also be important for the binding of the ⁇ -carboxylate group of amino acid nucleophiles.
- the binding of the C-terminal carboxylate group of peptide substrates is dependent on hydrogen bonds from the side chain of Asn51 and Glu145, the latter with the carboxylic acid group in its
- the transacylation reactions might be influenced by mutational replacements of Asn51 and Glu145 and possibly also, due to indirect effects, by replacement of Glu65.
- CPD-Y mutants with replacements at positions 51, 65 and 145 were investigated for their ability to catalyze transacylation reactions using H-Val-OH and H-Leu-OH as nucleophiles (Table II).
- Glu65 and Glu145 do not appear to be directly involved in the binding of the ⁇ -carboxylate group of amino acid nucleophiles at the pH where the reaction is carried out, they seem to exert an indirect influence such that their replacement affect the binding mode, as indicated by the elevated fa max values.
- amino acid amides are used as nucleophiles, the presence of the negatively charged Glu145 does not appear to exert a negative effect since the fa max values
- Asn51 was replaced with other amino acid residues and these enzymes were tested. Replacing Asn51 with Ser or Gin affected fa max in different directions. With N51S and H-Val-OH and H-Leu-OH as nucleophiles, fa max was 0.05 and 0.03, respectively (Table II). With N51Q, they were 0.97 and 0.96, respectively, and with the wild-type enzyme, fa max was 0.35 with both
- the k cat values for the hydrolysis of FA-Phe-Val-OH increase in the order N51Q ⁇ wild-type ⁇ N51S and this correlates inversely with fa max values with H-Val-OH as nucleophile which decrease in the order N51Q > wild type > N51S.
- carboxypeptidases exhibit no dependence on the nature of the leaving group. However, with peptide substrates where an amino acid acts as leaving group, this is not always the case. With carboxypeptidase Y, the most commonly employed serine carboxypeptidase, high yield of transpeptidation is only achieved when the leaving group is a hydrophilic amino acid. However, since
- carboxypeptidase Y in hydrolysis reactions exhibits a preference for hydrophobic amino acid leaving groups (P 1 ' amino acids), it would be beneficial, due to higher rate and specificity of the reaction, if such leaving groups were permissible in transpeptidation reactions as well. This would also permit modification of peptides and proteins, as isolated from natural sources, which presently are excluded due to hydrophobic C-terminal amino acid residues. The low yields due to the
- Mutants of carboxypeptidase Y were examined for the capacity to enhance yields of transacylation with substrates having leaving groups that are not hydrophilic. Some amino acid substitutions made in the active site of mutant carboxypeptidase Y enzymes were also made knowing they were not likely to improve the yields of transacylation reactions, but rather to investigate the mechanism of action of the leaving group dependence.
- Carboxypeptidase Y was obtained from Carlbiotech, Copenhagen, Denmark. All amino acids
- Hippuryl-L-Phe-OH, and Hippuryl-L- ⁇ -Penyllactic acid were purchased from Bachem, Switzerland. The
- Aminolysis reactions were carried out in the following way.
- the nucleophile was dissolved in 50 mM HEPES, 5 mM EDTA and pH was adjusted to 7.5, 5 ⁇ l substrate (8 mM FA-Ala-OBzl or FA-Ala-Xaa-OH in
- the reactant composition was determined by HPLC using a Waters HPLC equipped with a C-18 Waters Novapac 4 ⁇ reverse phase column and various gradients of acetonitrile in 0.1% trifluoroacetic acid.
- fraction of aminolysis ( fa) was expressed as the ratio between the formed aminolysis product and the sum of all products being formed, i.e. unconsumed substrate was disregarded in the
- a substrate acylates the essential serine residue which subsequently is deacylated by water, hence completing the hydrolysis reaction.
- an amine nucleophile e.g., an amino acid or amino acid amide
- the acyl-enzyme will be partitioned between water and the added amino component, in the latter case forming a new peptide bond (transpeptidation).
- the saturation curves can be described by equation (1).
- the upper limit of the fa termed fa max , is reached when N ⁇ K N,app .
- fa max can reach a maximum value of 1. This is, however, rarely obtained in practice.
- the concentration at which fa max /2 is reached, termed K N,app describes the affinity of the nucleophile for the enzyme and the dissociation of the aminolysis product.
- Peptide esters may function as substrates and, in this case, reaction with the amine component causes elongation of the peptide.
- the ratio of the hydrolysis to aminolysis reaction is not influenced by the nature of the alcohol leaving group, but that there is a pronounced effect of the nucleophile on fa max .
- fa max values ranging from 1.00 with H-Gly-NH 2 to 0.15 with H-Phe-OH as nucleophile are observed.
- nucleophiles are 0.87, 0.81, 0.90, 0.91, 0.32, 0.35, and 0.15, respectively, and thus similar to the fa max values obtained when these amino acids act as leaving groups (see Table IV).
- fa max contributes to low fa max values. While not meant to limit the invention, it is believed that the wide range of results with amino acids as leaving groups indicates that the magnitude of the rate constants which enter the expression for fa max are associated with the nature of the amino acid side-chain . It appears that the more hydrophobic the leaving group , the lower the fa max . A significant increase in fa max can be achieved by
- ⁇ -carboxylate group of the leaving group or nucleophile and the binding site for the C-terminal carboxylate group will influence fa max .
- N-terminal of the leaving group is a H 2 N- group
- Hippuryl-L- ⁇ -Phenyllactic acid ester bond
- N-terminal of the leaving group is a HO- group
- H-Gly-NH 2 as nucleophile
- the conformation in which an amino acid binds within the S 1 ' site may facilitate or restrain the access of water to the acyl component covalently attached to the essential serine residue (Serl46) due to the positioning of the amino group of the leaving group/nucleophile. It should, thus, be possible to achieve increased fa max values if the binding mode of a specific amino acid within the S 1 ' binding site is altered to prevent nucleophilic attack of water on the acyl-enzyme.
- N51Q exhibits increased fa max values in transpeptidation reactions is probably due to changes in the binding of amino acids within S 1 '.
- the mutation results in increased reaction with amine relative to water. This result would be consistent with a shorter distance between the acyl-enzyme and the ⁇ -amino group of the nucleophile/leaving group.
- carboxypeptidase Y (CPD-Y) has delineated the nature of the interaction between the C-terminal carboxylate group of the substrate and the enzyme. While not meant to be a limitation of the invention, it is believed that hydrogen bonds from the side chains of Asn51 and Glu145 appear to be responsible for the binding of the
- CPD-Y C-terminal carboxylate group of peptide substrates.
- the peptidase activity of CPD-Y is optimal at acidic pH.
- CPD-Y also catalyzes the release of amino acid amides from peptide amides but this activity is optimal at basic pH. It is likely that at the basic pH range Asn51 interacts with the carbonyl oxygen of the C-terminal carboxyamide group while Glu145 in its deprotonated (carboxylate) form interacts with the -NH 2 group of the substrate. Glu65 is hydrogen bonded to Asn51 and Glu 145 thereby orienting the two side chains involved in C-terminal recognition.
- Carboxypeptidase-Y was obtained from Carlbiotech, Copenhagen, Denmark.
- H-Val-OPr, H-Val-OBu, H-Val-NHCH 3 and H-Val-NHC 2 H 5 were from Peptech, Sydney, Australia. All other amino acids and amino acid
- mutants asn 51 ⁇ Gly, Cys and Val in the structural gene for CPD-Y and subsequent expression and purification of the mutants N51G and N51C were carried as described in
- Example 1 The purity of the enzymes was ascertained by SDS polyacrylamide gel electrophoresis. The preparation of the mutants N51A, N51D, N51T, N51Q, N51S, E145A and E145D has previously been described. The mutations Asn51 ⁇ Cys, Asn51 ⁇ Gly and Asn51 ⁇ Val were
- FA-Phe-Ala-OH and FA-Phe-Leu-OH were synthesized by standard methods. All enzymatic activities toward FA-substrates were determined spectrophotometrically at 329-337 nm using a Perkin Elmer lambda 7 or lambda 9
- Asn51 can be replaced with other hydrogen bond donors without impairing this wild-type enzyme.
- Asn51 ⁇ Cys mutation caused a significant increase in K N(app) and this is consistent with hydrogen bonds
- Glu145 and Glu65 appear to be much less important for the interaction with amino acid amide nucleophiles.
- the double mutant E65A-E145A was tested and it was found that K N(app) was drastically increased while fa max was somewhat reduced.
- K N(app) was drastically increased while fa max was somewhat reduced.
- the absence of both glutamic acids is detrimental to the binding of amino acid amides but the presence of one of them is sufficient to secure tight binding.
- Glu145 is negatively charged while Glu65 is uncharged and, accordingly,
- Glu145 is the one interacting with amino acid amides.
- the very low fa max and significantly elevated K N(app) obtained with E145Q confirm the significance of Glu145 in the interaction with amino acid amides.
- the C-terminus of peptide amides interact with the enzyme in an analogous way with the negatively charged Glu145 acting as a hydrogen bond acceptor. Since both Glu65 and Glu145 in the single mutants may function 'in this capacity there apparently may be some latitude concerning the length of the hydrogen bond. This is suggested by the fact that shifting the carboxylate group at position 145 one carbon atom away, i.e. E145D, has very small effects on both parameters.
- Valine N-methyl amide (H-Val-NH-CH 3 ) binds much less efficiently to CPD-Y than the unblocked H-Val-NH 2 .
- the fa max is almost as high (0.80).
- CPD-Y carboxypeptidase Y
- CPD-W carboxypeptidase
- the basic technique of the mutagenesis was in vitro DNA synthesis primed by mutagenic (degenerate) synthetic oligonucleotides using single-stranded
- the vps strain which requires leucine for growth, was used to search for desired mutants in a single direct screen.
- the transformed cells were plated on synthetic medium lacking leucine but containing one of various N-blocked-X-Leu-OH dipeptides. Only cells which express a protease that can release the terminal leucine can grown on these plates.
- the Vps + strain was used to screen for desired mutants in two steps. First, transformant colonies were overlaid by agar containing a chromogenic substrate which reveals CPD-Y activity. In the second step, the CPD-Y activity from the positive colonies was estimated towards different substrates in a chromogenic microtiter dish assay.
- CBZ-X-Leu-OH peptides and N-acetyl-L-alanine ⁇ -naphthyl ester were from Bachem; horseradish peroxidase type I, Crotalus atrox L-amino acid oxidase type VI, o-dianisidine and Fast Garnet Red GBC salt were from Sigma. Oligonucleotides were synthesized on an Applied Biosystems 394 DNA-RNA Synthesizer. LB, 2xYT and SOC medium were prepared according to Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY (1989). SC and YPD medium were prepared according to Sherman, Methods Enzymol., 194:3-21 (1991), with slight modifications (Tullin et al., Yeast, 7:933-941 (1991)).
- E. coli JM109 (recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi ⁇ (lac-proAB) F' [traD36 proAB + laqI q lacZ ⁇ M15]) (Yanisch-Perron et al., Gene, 33:103 (1985)); S. cerevisiae JHRY20-2C ⁇ 3 (MATa ⁇ prc1 leu2-3 leu2-112 ura3-52 his3- ⁇ 200 prcl- ⁇ 3::HIS3) (Blachly-Dyson et al., J. Cell Biol., 104:1183 (1987)); S.
- W2579 (MATa ⁇ prcl leu2-3 leu2-112 ura3-52 vpl1-1) (Nielsen et al. (1990) cited supra.).
- the vpl1 gene has been renamed vpsl (Robinson et al., Mol. Cell. Biol., 8:4936 (1988)).
- K2579LLR was isolated in the present study as a spontaneous mutant of W2579 that requires less leucine for growth.
- Bio-Rad Gene Pulser set at 25 ⁇ F, 200 ⁇ and 2.5 kV in
- E. coli JM109 transformed with pYSP32 was grown to an OD 600 of 0.5 in 2 ⁇ YT + 50 mg/l
- tetracycline One milliliter of this culture was superinfected with 20 ⁇ l of a >10 9 p.f.u./ml M13K07 helper phage stock in a 500 ml Erlenmeyer bottle. After incubation for 1 hour at 37°C, 200 ml 2 ⁇ YT + 50 mg/l tetracycline + 50 mg/l kanamycin was added. After incubation with agitation overnight at 37°C, ssDNA was purified by standard procedures (Sambrook et al., A
- Kanamycin selects for cells superinfected with helper phage M13K07.
- oligonucleotide Promega, Altered Sites Kit
- 80 ⁇ l 2 x annealing buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 50 mM NaCl
- 10 ⁇ l 10 x synthesis buffer 100 mM Tris-HCl pH 7.5, 5 mM of each of the four dNTPs, 10 mM ATP, 20 mM DTT
- 10 Weiss units T4 ligase New England Biolabs
- 20 units T4 DNA polymerase Promega
- DNA sequencing was performed by the Applied Biosystems dsDNA Taq DyeDeoxyTM terminator procedure for use with the Applied Biosystems Model 373A DNA
- Yeast strain K2579LLR was transformed with the mutated population of pYSP32 and plated on SC-ura-leu supplemented with 1.5 mM of a poor CBZ-X-Leu-OH peptide substrate as an enzyme-dependent leucine source.
- P 1 of the substrate was either Glu, Gly, Ser, His, Pro, Trp or Lys.
- the cells can grow on this medium only if they express proteases capable of releasing the C-terminal leucine.
- CPD-Y is not the only protease secreted by the cells that can catalyze this cleavage.
- Yeast strain JHRY20-2C ⁇ 3 was transformed with the mutated population of pYSP32 and plated on SC-ura plates, which were incubated at 30°C until colonies reached a diameter of 2-3 mm. Then each plate was overlaid with a fresh mixture of 3 ml 0.6% agar in water at 50°C and 2 ml dimethylformamide containing 0.25% AANE at room temperature. After incubation for 5 min at room temperature, 5 ml 0.4% Fast Garnet Red GBC salt in 10 mM sodium phosphate pH 7.0 buffer was poured on top; after 5 min incubation the plates were then rinsed in tap water. Colonies expressing active CPD-Y appear red, while those lacking vacuolar CPD-Y activity appear white (modified from Jones, 1977). Colonies expressing active CPD-Y were isolated by streaking onto SC-ura plates.
- the activity of each mutant was normalized to that of the wide type as follows. First, all absorption values were corrected by subtracting the background (absorption at same time point in wells without cells). The difference in correction absorption between two time points (corresponding to the amount of hydrolysis) was then normalized to account for variations in cell number, by dividing by the OD of the cells (OD of well with cells at time point 0 minus OD of well without cells at time point 0). Finally, the obtained activity estimate was divided by the corresponding estimate for the wild type. Time point 0 and 1 hour were used to calculate the relative activity towards CBZ-Phe-Leu-OH, while time points 0 and 16 hour were used to calculate the relative activity towards all other substrates. Isolation and kinetic characterization
- the mutant BamHI-BamHI PRC1 fragments of pYSP32 were inserted into the GAL expression vector pRA21 and introduced into yeast strain K2579LLR.
- the plasmid pRA21 is derived from p72UG (Nielsen et al. (1990) cited supra) by replacing the 918 bp BglII-SalI fragment with the 638 bp BglII-PvuII fragment of pWI3, thereby
- carboxypeptidase Y is predicted to be at most a marginal part of the S 1 binding site; it is in fact absent from the S 1 site.
- the model that used the crystal structure of wheat carboxypeptidase-W and the sequence of yeast carboxypeptidase Y yielded accurate guidance for
- oligonucleotides were designed relatively long (33 bases) to minimize biased annealing of oligonucleotides complementary to the introduced stop codons and
- Colonies of yeast strain JHRY20-2C ⁇ 3 transformed with DNA from all mutagenesis series were screened by this assay.
- mutagenesis series 1, half of the transformant colonies express active CPD-Y (Table VIII). If the mutagenesis event at each oligonucleotide target is independent of that of the other three targets, this number corresponds to a mutation frequency of 84% at each target. Of 10 s tested transformants from mutagenesis series 2, none had detectable CPD-Y activity. In this series, six codons were mutated simultaneously. Fewer codons were mutated in series 3-6 and 8, which yielded between 0.4 and 10% positive transformants, expressing a wide range of CDP-Y activities as indicated by the color intensities in the overlay assay. Mutagenesis series 7 yielded 50%
- transformants expressing active CPD-Y and the level of activity of all transformants was indistinguishable from that of the wild type transformants, suggesting that positions 215 and 216 can be varied with little effect on activity towards AANE.
- Transformants of yeast JHRY20-2C ⁇ 3 expressing active CPD-Y from mutagenesis series 3-8 were tested in this assay using an initial substrate concentration of 1 mM. Compared with the wild type, transformants from mutagenesis series 7 all exhibited activity levels around 100% towards all eight tested CBZ-X-Leu-OH substrates. Neither very low nor very high activity levels were observed, suggesting that positions 215 and 216 have little influence on P 1 preference.
- mutagenesis series 3 and 6 showed a broad range of activities with an average around 100%. Several mutants were fund with very low activity levels, just as several were found with very high activity levels towards one of the substrates, CBZ-Lys-Leu-OH. In one case the
- CPD-Y was purified from the mutants 178Ser and
- CPD-Y can catalyze the transpeptidation of proline insulin (INS-Pro-Lys-Ala-OH) to produce human insulin amide (INS-Pro-Lys-Thr-NH 2 ), and it has
- Mutants selected for the ability to hydrolyze a poor CBZ-X-Leu-OH peptide substrate as described herein can also be screened for the ability to
- NAME Mortensen, Uffe Hasbo
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- TCCTACGCC 9 INFORMATION FOR SEQ ID NO:10:
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- TCCNNNGCC 9 INFORMATION FOR SEQ ID NO: 29:
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
Abstract
Description
Claims
Priority Applications (4)
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EP95921432A EP0723585A1 (en) | 1993-10-28 | 1994-10-28 | Customized proteases |
NZ283995A NZ283995A (en) | 1993-10-28 | 1994-10-28 | Customized (mutant) carboxypeptidase Y having modified S1 subsite |
JP7517672A JPH09504438A (en) | 1993-10-28 | 1994-10-28 | Special protease with modified acyl transfer activity |
AU26513/95A AU679861B2 (en) | 1993-10-28 | 1994-10-28 | Customized proteases with altered transacylation activity |
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US14470493A | 1993-10-28 | 1993-10-28 | |
US08/144,704 | 1993-10-28 | ||
US08/329,892 US6187579B1 (en) | 1993-10-28 | 1994-10-27 | Customized proteases |
US08/329,892 | 1994-10-27 |
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US (2) | US6187579B1 (en) |
EP (1) | EP0723585A1 (en) |
JP (1) | JPH09504438A (en) |
AU (1) | AU679861B2 (en) |
CA (1) | CA2174525A1 (en) |
NZ (1) | NZ283995A (en) |
WO (1) | WO1995020039A2 (en) |
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- 1994-10-28 NZ NZ283995A patent/NZ283995A/en unknown
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US6586224B1 (en) | 1999-07-22 | 2003-07-01 | The Procter & Gamble Company | Subtilisin protease variants having amino acid deletions and substitutions in defined epitope regions |
US6562594B1 (en) | 1999-09-29 | 2003-05-13 | Diversa Corporation | Saturation mutagenesis in directed evolution |
CN1100789C (en) * | 2000-01-13 | 2003-02-05 | 中国人民解放军第二军医大学 | Preparation process of gene recomibination calcitonin or calcitonin analog |
WO2001081556A2 (en) * | 2000-04-24 | 2001-11-01 | The Procter & Gamble Company | Enzyme variants having one or more d-amino acid substitutions |
WO2001081556A3 (en) * | 2000-04-24 | 2002-07-18 | Procter & Gamble | Enzyme variants having one or more d-amino acid substitutions |
WO2006015879A1 (en) * | 2004-08-13 | 2006-02-16 | Roche Diagniostics Gmbh | C-terminal modification of polypeptides |
US8927230B2 (en) | 2004-08-13 | 2015-01-06 | Eucodis Bioscience Gmbh | C-terminal modification of polypeptides |
Also Published As
Publication number | Publication date |
---|---|
JPH09504438A (en) | 1997-05-06 |
US5945329A (en) | 1999-08-31 |
NZ283995A (en) | 2000-04-28 |
CA2174525A1 (en) | 1995-07-27 |
AU2651395A (en) | 1995-08-08 |
EP0723585A1 (en) | 1996-07-31 |
WO1995020039A3 (en) | 1996-03-21 |
US6187579B1 (en) | 2001-02-13 |
AU679861B2 (en) | 1997-07-10 |
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