A RAPID AND EFFECTIVE PROCEDURE FOR SCREENING PROTEASE MUTANTS

We describe a simple and effective procedure to screen for active prot eases among a large number of mutants. First, the mutants are genetica lly tested by the protease activity produced in the periplasm of trans formed bacteria which supplies the cells with a nitrogen source by hyd rolyzing a protein applied to plates, Then a less sensitive activity s taining and an X-ray film digestion assay are used to verify and estim ate the activity of the mutants that proved to be positive in the firs t step, Depending essentially on the level of periplasmic protease act ivity, the method can detect both the activity and the stability of th e expressed enzymes. We calibrated the method with transformants that produce wild-type trypsin, chymotrypsin and trypsin mutants of known a ctivity, Using this method we found two active revertants of the inact ive Asn102 trypsin mutant, by screening similar to 4.4X10(4) random mu tants that were generated by the polymerase chain reaction on a cDNA f ragment. This procedure should be useful in searching for proteases of novel specificity and/or reaction chemistry engineered by random muta genesis, and also for in vitro evolution studies.