Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae

Abstract

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.

Heterologous protein production using the Pichia pastoris expression system

“…This is best illustrated by the use of protease-deficient strains to improve the quality and yields of various heterologous proteins [111]. In general, the secreted recombinant proteins can potentially be proteolytically degraded in the culture medium by extracellular proteases, cell-bound proteases [57] and/or by intracellular proteases from lysed cells. P. pastoris extracellular proteases are not well documented, however, and this yeast reportedly secretes only low levels of endogenous proteins [18,53].…”

Enzymes for Wine Fermentation: Current and Perspective Applications

“…Interestingly, the same situation is undesirable for beer brewery as PrA degrades proteins (e.g., lipid transfer protein 1), necessary for foam formation. Apart from PrA, S. cerevisiae expresses different cell-bound proteases, some of which are not fully characterized [73].…”

Characteristics of Saccharomyces cerevisiae gal1Δ and gal1Δhxk2Δ mutants expressing recombinant proteins from the GAL promoter

“…The construction of strains carrying the deletion mutation of the GAL1, MIG1, and HXK2 genes is described below. The tc5:URA3:tc5 pop-out cassette used for gene deletion was obtained from pTcUR3 (Kang et al, 2000). The episomal HSA expression vector pYHSA5, which contains the HSA cDNA under the control of the GAL10 promoter, has been previously described (Kang et al, 1998).…”

“…The tc5:URA3:tc5 pop-out cassette used for gene deletion was obtained from pTcUR3 (Kang et al, 2000). The episomal HSA expression vector pYHSA5, which contains the HSA cDNA under the control of the GAL10 promoter, has been previously described (Kang et al, 1998). Briefly, the plasmid pYHSA5, an E. coli-yeast shuttle vector based on 2 A plasmid, was constructed by ligation of the 2.0 kb EcoRI/blunt-ended HindIII fragment containing the 5V untranslated region and the whole coding region of HSA with the 6.0 kb EcoRI/ blunt-ended SalI fragment obtained from YEGa;-HIR525 (Choi et al, 1994).…”

“…A secretory production system of the recombinant human serum albumin (HSA) has previously been developed, by ourselves, using the GAL10 promoter in S. cerevisiae (Kang et al, 1998(Kang et al, , 2000. In this study, a S. cerevisiae gal1Á strain, expressing HSA, was constructed using galactose as a gratuitous inducer.…”