PROTEINASE TRAPPING - SCREENING FOR VIRAL PROTEINASE MUTANTS BY ALPHA COMPLEMENTATION

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 alpha-fragment of beta-galactosidase and an active 2A proteinase of human rhinovirus 2 was expressed, alpha-complementation was not affected, as the 2A proteinase cleaved itself off the alpha-fragment. However, fusion of an inactive 2A prevented alpha-complementation, as the 2A polypeptide remained fused to the alpha-fragment. After random mutation of the 2A gene by PCR amplification, mutants were screened; M13 phage defective in a complementation were obtained at an efficiency of 5% and were shown to contain mutated 2A genes. Intermolecular cleavage was then examined by expressing an a fragment-inactive proteinase fusion protein as substrate for an active 2A proteinase expressed from an M13 vector. Alpha-complementation indicated intermolecular processing of the 2A cleavage site on the alpha-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.