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 alpha complementation were obtained at an efficiency of 5% and were shown to contain mutated 2A genes. Intermolecular cleavage was then examined by expressing an alpha 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.
Liebig
H D
HD
Institute of Biochemistry, University of Vienna, Austria.
Skern
T
T
Luderer
M
M
Sommergruber
W
W
Blaas
D
D
Kuechler
E
E
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002872:Chromosome Deletion; D010450:Endopeptidases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test / Q000379:methods*; D005822:Genetic Vectors; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012229:Rhinovirus / Q000201:enzymology / Q000235:genetics*; D014162:Transfection; D014780:Viruses / Q000201:enzymology / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D010450:Endopeptidases