Complete determination of disulfide forms of purified recombinant human serum albumin, secreted by the yeast Pichia pastoris.
In the case where the supply of material is limited from natural resources and/or risks of infection are to be avoided, recombinant proteins are an important substitute. Consequently, the physicochemical characterization of the primary and tertiary structures of such materials that are to be used clinically is indispensable. In this context, disulfide linkages play a significant structural role and their determination is of paramount importance. As the demand for human serum albumin (HSA), which contains 35 cysteine residues, is continually increasing, its industrial-scale production from the genetically engineered yeast Pichia pastoris is of interest. The present paper describes a methodology that allows the characterization of the multi-disulfide linkages including exact positions in purified recombinant HSA by use of gas-phase protein sequencing. Mild Edman degradation followed by isocratic analysis of the phenylthiohydantoin amino acids in combination with multienzymatic digestions in acidic conditions allowed the exact positions of the 17 disulfide bridges and 1 sulfhydryl group to be rigorously determined. The sulfhydryl content of the present recombinant HSA was the same as plasma HSA.
Ikegaya
K
K
Research Division, The Green Cross Corporation, Osaka, Japan.
Hirose
M
M
Ohmura
T
T
Nokihara
K
K
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010434:Pepsin A / Q000378:metabolism; D010669:Phenylthiohydantoin / Q000737:chemistry; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012709:Serum Albumin / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000737:chemistry
D004220:Disulfides; D011994:Recombinant Proteins; D012709:Serum Albumin; D013438:Sulfhydryl Compounds; D010669:Phenylthiohydantoin; D010434:Pepsin A; D003545:Cysteine