Anti-autolysis of trypsin by modification of autolytic site Arg117.
In order to improve the stability of trypsin, an approach to knock out the autolytic site has been carried out in this investigation. Compared with trypsins from other species, the autolytic site Arg117-Val118 of rat trypsin is the most interesting candidate to work on. The Arg117 residue was designed to be deleted or replaced by other amino acid residues to destroy the autolytic site. With DNA site-directed mutagenesis method, one deletion mutant and several replacement mutants were selected. After expression and purification, the kinetic and anti-autolytic properties of mutant trypsins were studied. No net charge difference of the trypsin molecules was observed by native PAGE analysis. Kinetic studies show that the activities of mutants vary from one another. R117L gives 32 times the activity of wild type trypsin while R117C has no detective activity. Among 8 selected mutants with characteristic properties, 7 of them give prolonged half life during anti-autolytic assay with the exception of R117M which is more sensitive to autolysis.
Li
X F
XF
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing, People's Republic of China.
Nie
X
X
Tang
J G
JG
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D051381:Rats; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics
D001120:Arginine; D014357:Trypsin