A new simplified procedure for C1 inhibitor purification. A novel use for jacalin-agarose.
C1 inhibitor (C1-INH), the major regulatory protein of the classical pathway of complement activation, is also involved in the regulation of several other plasma proteolytic systems including the coagulation, fibrinolytic and contact systems. All the previously published methods for the purification of C1-INH are time-consuming and some do not yield highly pure protein. Recently, it was reported that Jack fruit (Artocarpus integrifolia) lectin, also called jacalin, binds C1-INH. Since jacalin binds only a small number of human serum proteins it appeared that jacalin-agarose affinity chromatography would constitute a very selective early step for the purification of C1-INH. Consequently we have designed a new, simplified three-step procedure for the purification of C1-INH which includes PEG fractionation, jacalin-agarose chromatography and hydrophobic interaction chromatography on phenyl-Sepharose which takes advantage of the marked hydrophilicity of the inhibitor. This procedure has three major advantages over those which have been the most frequently used. Firstly, it includes only two fast chromatographic steps. Secondly, because the C1-INH pool is cleanly and predictably separated from the unwanted proteins by differential elution conditions in both chromatographic steps, no antigenic or functional assays are required to define the desired peaks. Thirdly, only the final product is dialyzed while all other methods required several buffer changes. For these reasons this procedure is much faster and simpler than the previously published methods. About 10-12 mg of highly purified and fully active C1-INH can be obtained within 1 day from 120 ml of plasma giving an average yield of 40-45%. This method may thus be highly adaptable to bulk purification for clinical use or for preparation of genetically or pathologically altered C1-INH from clinical specimens.
Pilatte
Y
Y
Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.
Hammer
C H
CH
Frank
M M
MM
Fries
L F
LF
D015153:Blotting, Western; D002845:Chromatography / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods*; D003174:Complement C1 Inactivator Proteins / Q000302:isolation & purification*; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D008970:Molecular Weight; D037121:Plant Lectins; D012995:Solubility
D003174:Complement C1 Inactivator Proteins; D037102:Lectins; D037121:Plant Lectins; C043696:jacalin