A nondenaturing preparative gel electrophoresis system for the recovery of functional proteins. Application to the identification of an endogenous protein inhibitor of fucosyl-transferase activities.
Electroelution of protein bands resolved by nondenaturing polyacrylamide gel electrophoresis was performed to identify an endogenous protein inhibitor of fucosyltransferase activities, called fuctinin, through its biological activity. After electrophoresis protein bands were negatively stained with zinc acetate, indicating that this staining technique can be also applied to nondenaturing polyacrylamide gels. However, even under appropriate electroelution conditions, a strong fucosyl-transferase inhibitory activity was eluted from the polyacrylamide gel itself that impeded the measure of fuctinin activity. As an alternative to electroelution, collection of proteins resolved by nondenaturing polyacrylamide gel electrophoresis as they are electrophoresed off the end of the gel was assayed. In the commercially available preparative electrophoretic systems, an elution chamber is limited by a semipermeable membrane on which proteins were adsorbed in the low-ionic-strength buffer used in nondenaturing electrophoresis. We demonstrate that the simple and inexpensive electrofractionation system described by Shain et al. (Anal. Biochem. 200, 47-51, 1992) can be successfully applied under nondenaturing conditions to purify and identify fuctinin through its biological activity. However, caution must be taken in the design of the apparatus in order to avoid local heating and thermal denaturation of proteins. The utility of this nondenaturing preparative electrophoresis system for the study of functional proteins is also demonstrated by the recovery of enzymatic activities of alkaline phosphatase and beta-galactosidase.
Ruggiero-Lopez
D
D
Department of Biochemistry, INSERM-CNRS 189, Lyon-Sud Medical School, Oullins, France.
Louisot
P
P
Martin
A
A
D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D004791:Enzyme Inhibitors / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors*; D011489:Protein Denaturation; D011506:Proteins / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate
D004791:Enzyme Inhibitors; D011506:Proteins; C089262:fuctinin; D012967:Sodium Dodecyl Sulfate; D005647:Fucosyltransferases