Intein-mediated protein ligation of expressed proteins
US6849428B1 · kind B1 · utility
Assignee
Inventors
Key dates
| Filing date | Feb 12, 1999 |
| Grant date | Feb 1, 2005 |
| Priority date | — |
| Expiry date | Feb 12, 2019 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC07K2319/92
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
A method for the ligation of expressed proteins which utilizes inteins, for example the RIR1 intein from Methanobacterium thermotrophicum, is provided. Constructs of the Mth RIR1 intein in which either the C-terminal asparagine or N-terminal cysteine of the intein are replaced with alanine enable the facile isolation of a protein with a specified N-terminal, for example, cysteine for use in the fusion of two or more expressed proteins. The method involves the steps of generating a C-terminal thioester-tagged target protein and a second target protein having a specified N-terminal via inteins, such as the modified Mth RIR1 intein, and ligating these proteins. A similar method for producing a cyclic or polymerized protein is provided. Modified inteins engineered to cleave at their C-terminus or N-terminus, respectively, and DNA and plasmids encoding these modified inteins are also provided.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.