Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Patent
1993-02-26
1995-04-04
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
435220, 435221, 435222, 4353201, 536 232, 935 10, 935 14, C12N 120, C12N 952, C12N 954, C12N 1500
Patent
active
054037379
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD OF THE INVENTION
The invention relates to serine protease variants derived from precursor serine proteases via recombinant and/or chemical methods to form protease variants having improved peptide ligase activity. The invention also includes novel ligation substrates which in combination with the serine protease variants and a second ligation substrate are capable Of forming a ligation product. The invention also relates to methods for forming such ligation products and the products formed thereby.
BACKGROUND OF THE INVENTION
Chemical approaches for synthesis and engineering of proteins offer many advantages to recombinant methods in that one can incorporate non-natural or selectively labelled amino acids. However, peptide synthesis is practically limited to small proteins (typically <50 residues) due to the accumulation of side-products and racemization that complicate product purification and decrease yields (for recent reviews see Kaiser, E. T. (1989) Acc. Chem. Res. 22, 47-54; Offord, R. E. (1987) Prot. Eng. 1, 151-157).
Proteolytic enzymes, in particular serine proteases, have reportedly been used as alternatives to synthetic peptide chemistry because of their stereoselective properties and mild reaction conditions (for reviews see Kullman, W. (1987) In: Enzymatic Peptide Synthesis, CRC Press, Florida U.S.; Chaiken, (1981) CRC Crit. Rev. Biochem. 11, 255-301). Such enzymes reportedly have been used to complement chemical coupling methods to produce larger peptides by blockwise enzymatic coupling of synthetic fragments. Inouye et al. (1979), J. Am. Chem. Soc., 101, 751-752 (insulin fragments); Hommandberg and Laskowski, (1979) Biochemistry 18, 586-592 (ribonuclease fragments)). However, the narrow substrate specificities and intrinsic hydrolytic (peptidase) activity of serine proteases have limited their use in peptide synthesis.
A central problem in the case of serine proteases in peptide synthesis is that hydrolysis of the acyl-enzyme intermediate is strongly favored over aminolysis (FIG. 1). Several laboratories have reported that the equilibrium is shifted from hydrolysis toward aminolysis by use of mixed or pure organic solvents to carry out catalysis (Coletti-Previero et al., (1969) J. Mol. Biol. 39, 493-501; Barbas et al., (1988) J. Am. Chem. Soc. 110, 5162-5166). However, enzymes are generally less stable and relatively insoluble in organic solvents (Wong et al., (1990) J. Am. Chem. Soc. 112, 945-953; Klibanov, (1986) Chemtech 16, 354-359). Further, kinetic activation barriers in organic solvents are higher for the charged transition-states involved leading to lower enzymatic activity. In an attempt to avoid these problems, one laboratory reported that thiolsubtilisin, a derivative of the bacterial serine protease in which the active site Ser221 was chemically converted to a Cys (S221C), shifted the preference for aminolysis to hydrolysis by >1000-fold for very small peptides. Nakasuta et al. (1987) J. Am. Chem. Soc. 109, 3808-3810.
This shift was attributed to the kinetic preference of thioesters to react with amines over water. Based upon similar principles, another laboratory reported that selenolsubtilisin had a 14,000-fold shift in preference for aminolysis over hydrolysis. Wu and Hilvert (1989) J. Am. Chem. Soc. 111, 4513-4514. However the catalytic efficiencies for aminolysis of a chemically activated ester by either thiol- or selenolsubtilisin are about 10.sup.3 - and 10.sup.4 -fold, respectively, below the esterase activity of wild-type subtilisin. Although chemically active esters have reportedly been used to increase the rates for acylation of thiol- or selenolsubtilisin (e.g. the acylation of thiolsubtilisin with a p-chlorophenyl ester of an 8-mer peptide for ligation with a 4-mer peptide in >50% DMF), such activated esters present synthetic difficulties as well as creating substrates prone to spontaneous hydrolysis in aqueous solvents (Nakatsuka et al. (1987) supra.).
The serine proteases comprise a diverse class of enzymes having a wide range of specif
REFERENCES:
patent: 4760025 (1988-07-01), Estell et al.
patent: 5155033 (1992-10-01), Estell et al.
Nakatsuka et al. J. Am. Chem. Soc. vol. 109, 1987, pp. 3808-3810.
Abrahmsen Lars B.
Burnier John
Wells James A.
Genentech Inc.
Hendricks Keith D.
Trecartin Richard F.
Wax Robert A.
LandOfFree
Serine protease variants having peptide ligase activity does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Serine protease variants having peptide ligase activity, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Serine protease variants having peptide ligase activity will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2380128