Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
1999-07-02
2003-01-28
Prouty, Rebecca E. (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C435S007600, C435S183000, C435S193000, C435S222000, C435S441000, C530S350000, C530S395000, C530S402000, C530S404000, C530S411000, C532S001000
Reexamination Certificate
active
06512098
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to chemically modified mutant proteins having modified glycosylation patterns with respect to a precursor protein from which they are derived. In particular, the present invention relates to a chemically modified mutant protein including a cysteine residue substituted for a residue other than cysteine in a precursor protein, the substituted cysteine residue being subsequently modified by reacting the cysteine residue with a glycosylated thiosulfonate. The present invention also relates to a method of producing the chemically modified mutant proteins and a glycosylated methanethiosulfonate. Another aspect of the present invention is a method of modifying the functional characteristics of a protein by reacting the protein with a glycosylated methanethiosulfonate reagent. The present invention also relates to methods of determining the structure-function relationships of chemically modified mutant proteins.
BACKGROUND OF THE INVENTION
Modifying enzyme properties by site-directed mutagenesis has been limited to natural amino acid replacements, although molecular biological strategies for overcoming this restriction have recently been derived (Cornish et al.,
Angew. Chem.,
Int. Ed. Engl., 34:621-633 (1995)). However, the latter procedures are difficult to apply in most laboratories. In contrast, controlled chemical modification of enzymes offers broad potential for facile and flexible modification of enzyme structure, thereby opening up extensive possibilities for controlled tailoring of enzyme specificity.
Changing enzyme properties by chemical modification has been explored previously, with the first report being in 1966 by the groups of Bender (Polgar et al.,
J. Am. Chem. Soc.,
88:3153-3154 (1966)) and Koshland (Neet et al.,
Proc. Natl. Acad. Sci. USA,
56:1606-1611 (1966)), who created a thiolsubtilisin by chemical transformation (CH
2
OH→CH
2
SH) of the active site serine residue of subtilisin BPN' to cysteine. Interest in chemically produced artificial enzymes, including some with synthetic potential, was renewed by Wu (Wu et al.,
J. Am. Chem. Soc.,
111:4514-4515 (1989); Bell et al.,
Biochemistry,
32:3754-3762 (1993)) and Peterson (Peterson et al.,
Biochemistry,
34:6616-6620 (1995)), and, more recently, Suckling (Suckling et al.,
Bioorg. Med. Chem. Lett.,
3:531-534 (1993)).
Enzymes are now widely accepted as useful catalysts in organic synthesis. However, natural, wild-type, enzymes can never hope to accept all structures of synthetic chemical interest, nor always be transformed stereospecifically into the desired enantiomerically pure materials needed for synthesis. This potential limitation on the synthetic applicabilities of enzymes has been recognized, and some progress has been made in altering their specificities in a controlled manner using the site-directed and random mutagenesis techniques of protein engineering. However, modifying enzyme properties by protein engineering is limited to making natural amino acid replacements, and molecular biological methods devised to overcome this restriction are not readily amenable to routine application or large scale synthesis. The generation of new specificities or activities obtained by chemical modification of enzymes has intrigued chemists for many years and continues to do so.
U.S. Pat. No. 5,208,158 to Bech et al. (“Bech”) describes chemically modified detergent enzymes where one or more methionines have been mutated into cysteines. The cysteines are subsequently modified in order to confer upon the enzyme improved stability towards oxidative agents. The claimed chemical modification is the replacement of the thiol hydrogen with C
1-6
alkyl.
Although Bech has described altering the oxidative stability of an enzyme through mutagenesis and chemical modification, it would also be desirable to develop one or more enzymes with altered properties such as activity, nucleophile specificity, substrate specificity, stereoselectivity, thermal stability, pH activity profile, and surface binding properties for use in, for example, detergents or organic synthesis. In particular, enzymes, such as subtilisins, tailored for peptide synthesis would be desirable. Enzymes useful for peptide synthesis have high esterase and low amidase activities. Generally, subtilisins do not meet these requirements and the improvement of the esterase to amidase selectivities of subtilisins would be desirable. However, previous attempts to tailor enzymes for peptide synthesis by lowering amidase activity have generally resulted in dramatic decreases in both esterase and amidase activities. Previous strategies for lowering the amidase activity include the use of water-miscible organic solvents (Barbas et al.,
J. Am. Chem. Soc.,
110:5162-5166 (1988); Wong et al.,
J. Am. Chem. Soc.,
112:945-953 (1990); and Sears et al.,
Biotechnol. Prog.,
12:423-433 (1996)) and site-directed mutagenesis (Abrahamsen et al.,
Biochemistry,
30:4151-4159 (1991); Bonneau et al., “Alteration of the Specificity of Subtilisin BPN' by Site-Directed Mutagenesis in its S
1
and S
1
′ Binding-Sites,”
J. Am. Chem. Soc.,
113:1026-1030 (1991); and Graycar et al.,
Ann. N. Y. Acad. Sci.,
67:71-79 (1992)). However, while the ratios of esterase-to-amidase activities were improved by these approaches, the absolute esterase activities were lowered concomitantly. Abrahamsen et al.,
Biochemistry,
30:4151-4159 (1991). Chemical modification techniques (Neet et al.,
Proc. Nat. Acad. Sci.,
56:1606 (1966); Polgar et al.,
J. Am. Chem. Soc.,
88:3153-3154 (1966); Wu et al.,
J. Am. Chem. Soc.,
111:4514-4515 (1989); and West et al.,
J. Am. Chem. Soc.,
112:5313-5320 (1990)), which permit the incorporation of unnatural amino acid moieties, have also been applied to improve esterase to amidase selectivity of subtilisins. For example, chemical conversion of the catalytic triad serine (Ser221) of subtilisin to cysteine (Neet et al.,
Proc. Nat. Acad. Sci.,
56:1606 (1966); Polgar et al.,
J. Am. Chem. Soc.,
88:3153-3154 (1966); and Nakatsukaet al.,
J. Am. Chem. Soc.,
109:3808-3810 (1987)) or to selenocysteine (Wu et al.,
J. Am. Chem. Soc.,
111:4514-4515 (1989)), and methylation of the catalytic triad histidine (His57) of chymotrypsin (West et al.,
J. Am. Chem. Soc.,
112:5313-5320 (1990)), effected substantial improvement in esterase-to-amidase selectivities. Unfortunately however, these modifications were again accompanied by 50- to 1000-fold decreases in absolute esterase activity.
Surface glycoproteins act as markers in cell-cell communication events that determine microbial virulence (Sharon et al.,
Essays Biochem.,
30:59-75 (1995)), inflammation (Lasky,
Annu. Rev. Biochem.,
64:113-139 (1995); Weis et al.,
Annu. Rev. Biochem.,
65:441-473 (1996)), and host immune responses (Varki,
Glycobiol.,
3:97-130 (1993); Dwek,
Chem. Rev.,
96:683-720 (1996)). In addition, the correct glycosylation of proteins is critical to their expression and folding (Helenius,
Mol. Biol. Cell,
5:253-265 (1994)) and increases their thermal and proteolytic stability (Opdenakker et al.,
FASEB J.,
7:1330-1337(1993)). Glycoproteins occur naturally in a number of forms (glycoforms) (Rademacher et al.,
Annu. Rev. Biochem.,
57:785-838 (1988)) that possess the same peptide backbone, but differ in both the nature and site of glycosylation. The differences exhibited (Rademacher et al.,
Annu. Rev. Biochem.,
57:785-838 (1988); Parekh et al.,
Biochem.,
28:7670-7679 (1989); Knight,
Biotechnol.,
7:35-40 (1989)) by each component within these microheterogeneous mixtures present regulatory difficulties (Liu,
Trends Biotechnol.,
10:114-120 (1992); Bill et al.,
Chem. Biol.,
3:145-149 (1996)) and problems in determining exact function. To explore these key properties, there is a pressing need for methods that will not only allow the preparation of pure glycosylated proteins, but will also allow the preparation of non-natural variants for the determination of structure-function relationships, such as structure-activity re
Davis Benjamin G.
Jones J. Bryan
Genencor International Inc.
McCutchen Doyle Brown & Enersen LLP
Prouty Rebecca E.
Rao Manjunath N.
Shuster Michael J.
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