A mutant trypsin-like enzyme from Streptomyces fradiae, created by site-directed mutagenesis, improves affinity chromatography for protein trypsin inhibitors.
The Ser-170 residue of a trypsin-like enzyme from Streptomyces fradiae (SFT), which is considered to be the active-site serine, was replaced with alanine by site-directed mutagenesis to improve the affinity chromatography step for a Kazal-type trypsin inhibitor pancreatic secretory trypsin inhibitor (PSTI). The resulting mutant SFT, designated as [S170A]SFT, was expressed in Streptomyces lividans and purified to homogeneity. [S170A]SFT was catalytically inactive, but still had the ability to bind tightly to PSTI and to soybean trypsin inhibitor with dissociation constants of 3.1 x 10(-7) M and 1.9 x 10(-8) M respectively. We further demonstrated that recombinant human PSTI secreted into Saccharomyces cerevisiae culture broth could be purified to homogeneity with a one-step [S170A]SFT-affinity column. The purified PSTI contained no molecules intramolecularly cleaved by active trypsin, which are found when trypsin-affinity chromatography is used for the purification. This eliminated the need for further separation of intact PSTI from intramolecularly cleaved PSTI by high-performance liquid chromatography, thus simplifying and improving its purification process.
Katoh
T
T
Shionogi Research Laboratories, Shionogi & Co. Ltd., Osaka, Japan.
Kikuchi
N
N
Nagata
K
K
Yoshida
N
N
D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*; D014357:Trypsin / Q000235:genetics / Q000378:metabolism; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000302:isolation & purification* / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D014359:Trypsin Inhibitor, Kazal Pancreatic; D014357:Trypsin