Simple polyacrylamide affinity gel electrophoresis using oleic acid for the isolation of chymotrypsin inhibitor.
Protease inhibitors have been usually isolated through a number of steps using various chromatographical methods, which are time consuming and tedious. In this report, an efficient and low-cost acrylamide affinity gel electrophoresis method for the detection and isolation of chymotrypsin inhibitor from a crude extract was studied. The affinity gel was obtained by immobilization of chymotrypsin on 5% (w/v) poly acrylamide-oleic acid gel, and the immobilized chymotrypsin showed high stability under varied concentrations of urea (0 to 8M), pH (4 to 10) and temperature (30 to 80 degrees C). The affinity gel made of immobilized chymotrypsin was applied to polyacrylamide affinity gel electrophoresis and reverse electrode electro-elution using a modified commercial electrophoresis kit. Polyacrylamide affinity gel electrophoresis method showed higher isolation efficiency for chymotrypsin inhibitor from Ganoderma lucidum crude extract than a chromatographical method. Specific activity and yield of chymotrypsin inhibitor increased around 2.3-folds and 1.4-folds, respectively, compared with a chromatographical method. Also, two isomers of the inhibitor could be isolated by this method. Therefore, this method can be applied for the detection and isolation of bio-active molecules as a fast and economical method.
Lim
Jin Ik
JI
National Core Research Center for Nanomedical Technology, Yonsei University, 262 Seongsanno, Seodaemun-gu, Seoul, 120-749, Republic of Korea. limjinik@hanmail.net
Lim
Kook-Jin
KJ
Na
Yun-Cheol
YC
Lee
Yong-Keun
YK
D002846:Chromatography, Affinity / Q000379:methods*; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D019301:Oleic Acid / Q000737:chemistry*; D011480:Protease Inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D020940:Reishi / Q000737:chemistry*
D011480:Protease Inhibitors; D019301:Oleic Acid; D002918:Chymotrypsin