Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae.
In order to direct the persistent expression of recombinant human serum albumin (HSA) from the GAL10 promoter in the yeast Saccharomyces cerevisiae, we carried out periodic feeding of galactose during shake-flask cultures. Unexpectedly, the recombinant protein secreted was observed to undergo rapid degradation, which was apparently accelerated by carbon-source feeding. The extracellular degradation of HSA occurred even in the strain deficient in the major vacuolar proteases PrA and PrB, and in the strain lacking the acidic protease Yap3p (involved in the generation of HSA-truncated fragments). Interestingly, the degradation correlated closely with the acidification of extracellular pH and thus was significantly overcome either by buffering the culture medium above pH 5.0 or by adding amino acid-rich supplements to the culture medium, which could prevent the acidification of medium pH during cultivation. Addition of arginine or ammonium salt also substantially minimized the degradation of HSA, even without buffering. The extracellular degradation activity was not detected in the cell-free culture supernatant but was found to be associated with intact cells. The results of the present study strongly suggest that the HSA secreted in S. cerevisiae is highly susceptible to the pH-dependent proteolysis mediated by cell-bound protease(s) whose activity and expression are greatly affected by the composition of the medium.
Kang
H A
HA
Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon.
Choi
E S
ES
Hong
W K
WK
Kim
J Y
JY
Ko
S M
SM
Sohn
J H
JH
Rhee
S K
SK
D003470:Culture Media; D005690:Galactose / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009584:Nitrogen / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D012709:Serum Albumin / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
D003470:Culture Media; D011994:Recombinant Proteins; D012709:Serum Albumin; D010447:Peptide Hydrolases; D009584:Nitrogen; D005690:Galactose