Development of a high-level expression system for deuterium-labeled human serum albumin.
We have developed an expression system for recombinant human serum albumin (rHSA) using methylotrophic yeast Pichia pastoris Mut(+) transformants together with the multiple cross-over integration of the vector containing human serum albumin (HSA). After 86 h of methanol induction, the secreted rHSA reached levels of approximately 320 mg/l in 100% H(2)O medium and approximately 180 mg/l in 70% D(2)O/30% H(2)O (v/v) medium in a fed-batch fermenter. The structures of the obtained rHSA and plasma-derived HSA (pHSA) were virtually identical as viewed from various physicochemical techniques such as HPLC, SDS gel electrophoresis, and CD. NMR peaks of the partially deuterium (D)-labeled rHSA (DrHSA) were quite sharp compared to those of pHSA due to suppression of the intramolecular nuclear Overhauser effect, promising further structural studies of the whole HSA molecule in the solution state using the recent NMR techniques.
Tomida
Mihoko
M
Department of Biochemistry, Gifu University School of Medicine, Gifu, 500-8705 Japan.
Kimura
Masashi
M
Kuwata
Kazuo
K
Hayashi
Tomoya
T
Okano
Yukio
Y
Era
Seiichi
S
D003903:Deuterium; D005786:Gene Expression Regulation; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008279:Magnetic Resonance Imaging / Q000379:methods; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012709:Serum Albumin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D012709:Serum Albumin; D003903:Deuterium