Subtilisin variants capable of cleaving substrates containing ba

Details Subtilisin variants capable of cleaving substrates containing ba Subtilisin variants capable of cleaving substrates containing ba

1995-07-19

1998-11-17

Grimes, Eric

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

435 681, 435 691, 4351723, 435221, 4352523, 43525231, 4353201, 536 232, C12P 2106, C12N 954, C12N 1557, C12N 1575

Patent

active

058375163

ABSTRACT:
The bacterial serine protease, subtilisin BPN', has been mutated so that it will efficiently and selectively cleave substrates containing basic residues. Combination mutants, where Asn 62 was changed to Asp, Gly 166 was changed to Asp (N62D/G166D), and optionally Tyr 104 was changed to Asp had a larger than additive shift in specificity toward substrates containing basic residues. Suitable substrates of the variant subtilisin were revealed by sorting a library of phage particles (substrate phage) containing five contiguous randomized residues. This method identified a particularly good substrate, Asn-Leu-Met-Arg-Lys- (SEQ ID NO: 35), that was selectively cleaved in the context of a fusion protein by the N62D/G166D subtilisin variant. A particularly good substrate for N62D/G166D/Y104D would be Asn-Arg-Met-Arg-Lys- (SEQ ID NO: 76). Accordingly, these variant subtilisin are useful for cleaving fusion proteins with basic substrate linkers and processing hormones or other proteins (in vitro or in vivo) that contain basic cleavage sites.

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