Erschienen:
National Academy of Sciences of the United States of America, 1991
Erschienen in:
Proceedings of the National Academy of Sciences of the United States of America, 88 (1991) 14, Seite 5979-5983
Sprache:
Englisch
ISSN:
0027-8424
Entstehung:
Anmerkungen:
Beschreibung:
<p>Many virally encoded proteinases cleave themselves out of a polyprotein, with cleavage occurring usually at their own N terminus. This property was used to develop an in vivo screening system using the lacZ gene fragment of M13mp18. When a fusion protein of the α fragment of β-galactosidase and an active 2A proteinase of human rhinovirus 2 was expressed, α complementation was not affected, as the 2A proteinase cleaved itself off the α fragment. However, fusion of an inactive 2A prevented α complementation, as the 2A polypeptide remained fused to the α fragment. After random mutation of the 2A gene by PCR amplification, mutants were screened; M13 phage defective in α complementation were obtained at an efficiency of 5% and were shown to contain mutated 2A genes. Intermolecular cleavage was then examined by expressing an α fragment-inactive proteinase fusion protein as substrate for an active 2A proteinase expressed from an M13 vector. α complementation indicated intermolecular processing of the 2A cleavage site on the α fragment-inactive proteinase fusion protein. This versatile system thus allows the high-density screening of both active and inactive proteinase mutants, cleaving either intramolecularly or intermolecularly, and should be applicable to other proteinases of high specificity.</p>