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pubmed:abstractText |
The complete amino acid sequence of human plasma apolipoprotein C-II (apoC-II) has been synthesized chemically by the solid-phase method using phenylacetamidomethyl-resin. All amino acids were coupled to the peptide-resin in the presence of 1-hydroxybenzotriazole; tert-butyloxycarbonyl-protected amino acids with the appropriate side-chain-protecting groups that are stable to the reaction conditions used in the solid-phase methodology were used. After cleavage and deprotection, the crude apoC-II was purified by ion-exchange chromatography and then by reverse-phase high-performance liquid chromatography. The purified apolipoprotein was found to elute as a single peak under various chromatographic conditions, and the overall yield of the final purified protein was 20.7%. Synthetic apoC-II was characterized by several complementary analytical techniques including amino acid composition, Edman phenylisothiocyanate degradation, polyacrylamide gel electrophoresis, and high-performance liquid chromatography. The final product was found to be homogeneous and to activate normal human post-heparin lipase to the same extent as native apoC-II. The synthetic protein is also equally immunoreactive as native apoC-II.
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