C‐Terminal Proteolytic Degradation of Recombinant Desulfato‐Hirudin and Its Mutants in the Yeast Saccharomyces cerevisiae

Abstract

The potent thrombin inhibitor hirudin variant 1, originally isolated from the leech Hirudo medicinalis, was expressed in Saccharomyces cerevisiae under the control of a truncated glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter fragment. Fusion of the yeast acid phosphatase (PH0.5) signal sequence to the hirudin gene led to quantitative secretion of recombinant desulfato-hirudin variant 1 (r-hirudin) into the extracellular medium in a growth-dependent manner. In comparison to the genuine molecule, r-hirudin lacks the sulfate group at the Tyr in position 63. Besides the fulllength protein of 65 amino acids (hir65), chemical analysis revealed the presence mainly of two derivatives lacking the last amino acid Gln (hir64) or the penultimate Leu (hir63) in addition. When expressing r-hirudin in mutant strains defective in all but one of the three major known carboxypeptidases, it turned out that the vacuolar carboxypeptidase yscY as well as the a-factor precursor-processing carboxypeptidase, ysca, participate in the C-terminal degradation of r-hirudin. Direct involvement of yscY and ysca was confirmed by sequential disruption of their structural genes PRCl and KEXl, respectively. Disruption of PRAl, coding for the yscY-processing proteinase yscA, also abolished yscY-mediated C-terminal r-hirudin degradation, but clearly reduced the overall expression yield. Since ysca is described to be highly specific for basic amino acids which are not present at the C-terminus of r-hirudin, a series of r-hirudin mutants with changes in the C-terminal amino acids were constructed and analysed for ysca-mediated and yscY-mediated degradation. Chromatographic analysis of the expression products confirmed the preference of ysca for basic amino acids, although Tyr, Leu and Gln were also hydrolysed. It could further be concluded that ysca might also be responsible for the C-terminal degradation of recombinant atrial natriuretic factor and epidermal growth factor expressed in yeast.Saccharomyces cerevisiae has, over the years, gained wide-spread importance as a microbial host for the expression of heterologous proteins. Baker's yeast turned out to be especially valuable if secretion of the heterologous protein into the medium and correct native folding of the expression product were required (for review, see Romanos et al., 1992). In most cases, hybrid genes between the prepro-part (leader) of the a-factor precursor (MFal) and the coding region of the foreign protein have been constructed. Despite many reports on the successful use of the a-factor leader directed secretion in yeast, this system is often hampered by incomplete processing, only partial secretion, N-terminal heterogeneity and, consequently, rather low production yields of the genuine molecules (Brake, 1989).

Purification, Characterization and Biological Evaluation of Recombinant Leech‐Derived Tryptase Inhibitor (rLDTI) Expressed at High Level in the Yeast Saccharomyces Cerevisiae

“…As described above, putative C-terminally truncated byproducts of rLDTI were observed during purification. This phenomenon has been described for other heterologous proteins expressed in S. cerevisiae and has, in the case of recombinant hirudin, been assigned to the action of yeast carboxypeptidases (Nguyen et al, 1991 ;Heim et al, 1994). In order to elucidate the nature of the C-terminal truncation of rLDT1 and to identify the proteases involved in this process, the generation of the C-terminally truncated byproducts was followed in a series of strains isogenic with host strain H449 but deficient in either carboxypeptidase yscY (TR1376) or ysca (TR1417) or both of them (TR1456).…”

“…Shake-flask fermentations of yeast transformants using minimal medium for pre-culturing and complex HE40 or semi-synthetic HE42 as main culture media were carried out as described in Heim et al (1994), except that CuSO, was generally added to the main culture at a final concentration of 1 mM to induce the CUPl promoter (Hottiger et al, 1994).…”

Recombinant hirustasin: Production in yeast, crystallization, and interaction with serine proteases

“…The latter plasmid carried the hirustasin expression cassette in counter-clockwise orientation with respect to the dLeu2 marker. Saccharomyces cerevisiae strain TR1456 (MATa, prbl-I, cpsl-3, leu2-3,112, ura3A5, kexl::ura3A5, prcl::ura3A5; [cir"]; Heim et al, 1994) was transformed with the expression plasmid pHE171R according to Klebe et al (1983).…”

Characterization of the microheterogeneities of PIXY321, a genetically engineered granulocyte‐macrophage colony‐stimulating factor/interleukin‐3 fusion protein expressed in yeast

“…By analogy, the involvement of of a full-length molecule to a certain extent, the last residue of PIXY321 is totally missing. Using yeast mutant strains defective Gly180 in a similar fifth helix in PIXY321 is likely to impede the conformational flexibility and the formation of the cyclic for various proteases, Heim et al [34] have proved that the hirudin C-terminal heterogeneity is the result of the action of two imide intermediate required in the deamidation reaction mechanism [44]. proteases, the vacuolar carboxypeptidase yscY and the A-factor processing carboxypeptidase ysc A.…”