High-yield production and characterization of biologically active recombinant aprotinin expressed in Saccharomyces cerevisiae.
Aprotinin is a polypeptide composed of 58 amino acid residues and has a molecular weight of 6512Da. The 58 amino acid residues are arranged in a single polypeptide chain, which is cross-linked by three disulfide bridges and folded to form a pear-shaped molecule. To express recombinant aprotinin in Saccharomyces cerevisiae, a synthetic gene encoding aprotinin was constructed and fused in frame with the pre-sequence of the S. cerevisiae MATalpha1 gene at the cleavage site of signal peptidase. The expression of aprotinin in S. cerevisiae was carried out using the PRB1 promoter. Aprotinin was secreted as a biologically active protein at a concentration of 426 mg/L into high cell density fermentation medium of 70.9 g/L cell dry weight. The purification process consisted of only three major steps and provided consistent yields of recombinant aprotinin using gel filtration high-pressure liquid chromatographic (HPLC) with a purity level higher than 99% and was free of non-aprotinin-related impurities. The recombinant aprotinin had the same characteristics as bovine aprotinin in a number of analytical methods, including alpha2-plasmin inhibition assay, amino acid composition, N-terminal amino acid sequence determination, and mass spectrum analysis. With further optimization of the purification process and culture conditions for high-yield production by S. cerevisiae, this source of recombinant aprotinin may be a promising approach for the commercial manufacture of aprotinin for pharmaceutical use instead of bovine aprotinin.
Meta
Akihiro
A
Research Department 1, The Chemo-Sero-Therapeutic Research Institute, 1314-1 Kyokushi-Kawabe, Kikuchi, Kumamoto, Japan. meta@kaketsuken.or.jp
Nakatake
Hiroshi
H
Imamura
Takayuki
T
Nozaki
Chikateru
C
Sugimura
Kazuhisa
K
D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D002417:Cattle; D015966:Gene Expression Regulation, Fungal; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D007611:Aprotinin