Streptomyces erythraeus trypsin for proteomics applications.
Among trypsin family proteases, bovine and porcine trypsins are currently the enzymes of choice for proteomics applications. However, there are trypsins from other sources that have higher catalytic activities than mammalian trypsins. Of these, Streptomyces erythraeus trypsin (SET) is particularly attractive, because SET has more than 1 order of magnitude greater amidase activity than mammalian trypsin and is resistant to autolytic degradation. These properties are advantageous for many proteomics applications. To evaluate this protease for proteomic applications, we expressed SET in E. coli, purified it to homogeneity, and then examined its enzymatic properties. As expected, recombinant SET (rSET) had greater than an order of magnitude higher amide bond hydrolysis activity (Km/k(cat)) for both N(alpha)-benzoyl-L-arginine-p-nitroanilide and N(alpha)-benzoyl-L-lysine-p-nitroanilide than modified porcine trypsin and did not show any sign of autolytic degradation after 96 h of incubation at 37 degrees C. The performance of rSET for proteomic applications was evaluated by applying the protease for in-solution and in-gel digestion of bovine serum albumin, and for 18O labeling of peptides. These results confirmed that rSET has the potential to be a useful protease in such proteomic experiments. We also report various properties of rSET that are fundamental to the use of this protease for proteomics applications.
Kiser
Jianying Z
JZ
Case Center for Proteomics and Bioinformatics, Department of Pharmacology, Department of Ophthalmology and Visual Sciences, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio, USA.
Post
Marc
M
Wang
Benlian
B
Miyagi
Masaru
M
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods*; D016930:Saccharopolyspora / Q000201:enzymology*; D013379:Substrate Specificity; D013552:Swine; D014357:Trypsin / Q000378:metabolism*
D001426:Bacterial Proteins; D014357:Trypsin