Enhanced proteolytic cleavage of recombinant fusion proteins
US5416007A · kind A · utility
Assignee
Inventors
Key dates
| Filing date | Jul 12, 1994 |
| Grant date | May 16, 1995 |
| Priority date | — |
| Expiry date | Jul 12, 2014 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC07K2319/75
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
Disclosed are methods for increasing the yield of intact target proteins by cleaving fused polypeptides made by recombinant DNA techniques. The fused polypeptides are designed at the DNA level to have a preselected primary cleavage site in a pendant polypeptide fused to a protein of interest. Structural features of the fused polypeptide and cleavage reaction environment are controlled to favor cleavage by a preselected cleavage agent at the primary cleavage site over a second cleavage agent-sensitive amino acid sequence in the target protein. The cleavage reaction is terminated before completion when the ratio of intact target protein to truncated, cleaved target protein is optimized, and the remaining reaction mixture comprising uncleaved fused polypeptide is resubjected to the cleavage agent. The presence of charged organic molecules in the cleavage reaction mixture favors cleavage at the primary cleavage site. The endopeptidase used for cleavage may be immobilized on an insoluble support matrix.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.