Structure-function analysis of plasma cholesteryl ester transfer protein by protease digestion and expression of cDNA fragments in Escherichia coli.

摘要: In an attempt to define active domain of the protein, fragments cholesteryl ester transfer protein (CETP) were obtained by limited digestion native, plasma-derived with trypsin, chymotrypsin, or Staphylococcus aureus V8 protease expression CETP cDNA restriction in Escherichia coli. Although native these proteases resulted extensive fragmentation and loss intact 74-kDa molecule as shown sodium dodecyl sulfate-polyacrylamide gel electrophoresis, CE activity was unaffected (trypsin chymotrypsin treatment) only partially lost (V8 treatment). Analysis molecular sieve chromatography showed that transfer-active product this proteolysis consisted polypeptide which remained associated, retaining weight CETP. These proteolyzed complexes resistant dissociation dithiothreitol, 8 M urea, delipidating agents. As activity, found possess a stable conformation unchanged buffers containing up 4.5 following exposure even higher (8 M) urea concentrations. polypeptides from bacterially expressed be catalytically inactive although they contained epitope for inhibitory anti-CETP monoclonal antibody had emulsion binding properties similar Selected synthetic peptides (including peptide epitope) also devoid activity. Thus, no evidence independently subunit Together, results indicate possesses distinct highly tertiary structure is required catalytic