Subtilisin variants capable of cleaving substrates containing dibasic residues
US5780285A · kind A · utility
Assignee
Inventors
Key dates
| Filing date | Mar 3, 1995 |
| Grant date | Jul 14, 1998 |
| Priority date | — |
| Expiry date | Mar 3, 2015 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC07K2319/75
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
The bacterial serine protease, subtilisin BPN', has been mutated so that it will efficiently and selectively cleave substrates containing dibasic residues. A combination mutant, where Asn 62 was changed to Asp and Gly 166 was changed to Asp (N62D/G166D), had a larger than additive shift in specificity toward dibasic substrates. 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-, that was selectively cleaved in the context of a fusion protein by the N62D/G166D subtilisin variant. Accordingly, this variant subtilisin may be useful for cleaving fusion proteins with dibasic substrate linkers and processing hormones or other proteins (in vitro or in vivo) that contain dibasic cleavage sites.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.