[Structure of recombinant human serum albumin from Pichia pastoris].
The structure of recombinant human serum albumin derived from Pichia pastoris (rHSA) was analyzed in detail. Complete amino acid analysis was performed by the phenyl isothiocyanate precolumn labeling method. The amino terminal sequence was determined by the Edman degradation. The carboxyl terminal amino acid was determined by digestion with carboxypeptidase, and the carboxyl terminal peptide fragment was analyzed by electrospray mass spectrometry. The peptide fragments of rHSA digested with Lysylendopeptidase, Endoproteinase Glu-C, or Endoproteinase Asp-N were analyzed by electrospray mass spectrometry. The complete amino acid composition, the terminal sequences and the complete amino acid sequence of rHSA agreed with the primary structure deduced from its cDNA. The elution pattern of reduced and carboxymethylated rHSA digested with Lysylendopeptidase and the elution pattern of intact rHSA digested with pepsin were respectively similar to those of plasma-derived human serum albumin (pHSA). The pattern of CD spectrum of rHSA was identical in both shape and magnitude to that of pHSA. 1H-NMR spectra of rHSA and pHSA in deuterium oxide showed the same signal patterns in the observed region (delta 10.5-0.5 ppm). Cross peaks assigned to the alpha proton-beta proton of Asp-1 (delta 4.2/2.8 ppm) and the delta proton-epsilon proton of lysine residues (delta 2.8-3.2/1.4-2.0) showed the same cross peak patterns and chemical shifts in two-dimensional phase sensitive double-quantum filtered 1H-1H correlation spectra of rHSA and pHSA.
Ohtani
W
W
Research Division, Green Cross Corporation, Osaka, Japan.
Masaki
A
A
Ikeda
Y
Y
Hirose
M
M
Chuganji
M
M
Takeshima
K
K
Kondo
M
M
Sumi
A
A
Ohmura
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010843:Pichia; D011994:Recombinant Proteins / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry*
D000596:Amino Acids; D011994:Recombinant Proteins; D012709:Serum Albumin