Isolation and purification of human type I procollagen by adsorption to glass beads

Abstract

Procollagen from the culture medium of human foreskin fibroblasts is efficiently adsorbed on controlled-pore glass (CPG) beads. Elution of adsorbed protease(s), capable of procollagen degradation, is accomplished with 1 m phosphate. This allows subsequent purification steps to be accomplished without detectable degradation of the high-molecular-weight procollagen form which is subsequently eluted with 1 m Tris. Analysis of the Tris elution fraction from CPG beads by sodium dodecyl sulfate-agarose-polyacrylamide electrophoresis on 2% gels indicated that the majority of protein is types I and III procollagens and partially processed intermediates. Types I and III procollagens were separated by DEAE-cellulose chromatography, and presumptive undegraded type I procollagen was resolved from processed forms by molecular sieve chromatography in 1 m CaCl 2 on agarose beads. The high-molecular-weight type I human procollagen isolated by this method was found to contain both amino and carboxy-terminal propeptides. Two α1 and one α2 procollagen chains, disulfide bonded via the carboxy-terminal propeptides, are present per molecule. This procedure represents an efficient and relatively rapid method for preparing human procollagen in sufficient quantity for detailed chemical analysis.