Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1994-10-27
2001-02-13
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S068100, C435S069100, C435S212000, C435S219000, C435S226000, C435S252300, C435S471000, C536S023200
Reexamination Certificate
active
06187579
ABSTRACT:
BACKGROUND OF THE INVENTION
Peptides used for pharmaceutical purposes will, in the future, more frequently be produced through the exploitation of genetic engineering. However, genetic engineering has limits to its capabilities. For example, expression of recombinant peptides bearing non-naturally occurring L-amino acids, D-amino acids, radioactive amino acids, and other detectable labels is not possible through recombinant techniques because there is no genetic code which codes for these modifications. In addition, naturally occurring amino acid modifications such as C-terminal amide group substitution, which are routinely performed in vivo, are difficult to perform in vitro. These post-translation modifications are important because they often result in the most potent or longest acting form of the peptide and constitute the naturally occurring form of the peptide often needed for pharmaceutical use.
There are techniques for modification of recombinant peptides. One such technique is C-terminal &agr;-carboxyl amidation, as described by Bongers et al.,
Int. J. Peptide Protein Res.,
40:268 (1992) utilizing an &agr; amidating enzyme as described in Henriksen et al.,
J. Am. Chem. Soc.,
114:1876-1877 (1992); and Ohsuye et al.,
Biochem. Biophys. Res. Commun.,
150:1275-1281 (1988). However, these techniques are limited to those modifications for which there exists a natural enzyme or chemical method capable of performing the desired modification.
Amidation of peptides has been performed through protease catalyzed replacement reactions (transpeptidation) using an amino acid amide or peptide amide as a nucleophile. Sahina et al.,
Chem. Pharm. Bull.,
36:4345-4354 (1988); Sahina et al.,
Chem. Pharm. Bull.,
37:811-812 (1989); Breddam et al.,
J. Peptide Protein Res.,
37:153-160 (1991). Yields using these techniques are typically quite low. However, transpeptidation reactions catalyzed by serine or thiol-proteases, under appropriate reaction conditions, have been carried out in high yields. Breddam et al. (1991) cited supra. Although protease catalyzed 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.
Hence, there is a need to provide 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 naturally occurring protease enzyme.
SUMMARY OF THE INVENTION
These and other objectives are provided for by the method of the invention. 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 transacylation. The preferred preselected substrates are peptides having an acidic or basic amino acid at the penultimate position. The preferred preselected 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, modifying at least one codon for at least one amino acid in the active site to form a mutant DNA sequence, and transforming a suitable host cell with the mutant DNA to provide for expression of the customized protease. In a preferred version, 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. The targeted site preferably includes a codon for an amino acid in the active site which is replaced by the stop codon. 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 catalyzed by the customized protease is preferably not substantially transacylated by the known enzyme. 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 modification of recombinant peptides. The transacylation products produced are modified by substitution at the C-terminal or N-terminal end with various 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 invention.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides customized proteases (i.e., 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 capability to perform transacylation reactions not substantially catalyzed by the known protease (i.e. yields less than 10%) or the capability to perform transacylation reaction with improved or enhanced yields (i.e., 80 to 100%) or both. The mutant customized proteases have been modified so that the protease can perform transacylation reactions with different preselected substrates and/or different preselected nucleophiles than the known protease. The 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 preselected substrates and/or nucleophiles. The customized protease can also exhibit enhanced or improved yields of transacylation. Preselected substrates are preferably those that have an acidic or basic penultimate amino acid. Preselected nucleophiles are preferably acidic or basic amino acid amides.
The present disclosure will utilize the following terminology. This terminology is
Breddam Klaus
Kielland-Brandt Morten C.
Mortensen Uffe Hasbo
Olesen Kjeld Ove
Stennicke Henning Ralf
Achutamurthy Ponnathapu
Carlsberg A/S
Foley & Lardner
Moore William W.
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