Complete Determination of Disulfide Forms of Purified Recombinant Human Serum Albumin, Secreted by the Yeast Pichia pastoris

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• 作者：Kazuo Ikegaya ; Masaaki Hirose ; Takao Ohmura ; and Kiyoshi Nokihara
• 刊名：Analytical Chemistry
• 出版年：1997
• 出版时间：June 1, 1997
• 年：1997
• 卷：69
• 期：11
• 页码：1986 - 1991
• 全文大小：224K
• 年卷期：v.69,no.11(June 1, 1997)
• ISSN：1520-6882

文摘

In the case where the supply of material is limited fromnatural resources and/or risks of infection are to beavoided, recombinant proteins are an important substitute. Consequently, the physicochemicalcharacterizationof the primary and tertiary structures of such materialsthat are to be used clinically is indispensable. Inthiscontext, disulfide linkages play a significant structuralroleand their determination is of paramount importance.Asthe demand for human serum albumin (HSA), whichcontains 35 cysteine residues, is continually increasing,its industrial-scale production from the genetically engineered yeast Pichia pastoris is of interest.The presentpaper describes a methodology that allows the characterization of the multi-disulfide linkages including exactpositions in purified recombinant HSA by use of gas-phaseprotein sequencing. Mild Edman degradation followedby isocratic analysis of the phenylthiohydantoin aminoacids in combination with multienzymatic digestions inacidic conditions allowed the exact positions of the 17disulfide bridges and 1 sulfhydryl group to be rigorouslydetermined. The sulfhydryl content of the presentrecombinant HSA was the same as plasma HSA.