A novel yeast expression/secretion system for the recombinant plant thiol endoprotease propapain

Abstract

A new high-yield yeast expression/secretion system has been adapted for the plant thiol endoprotease papain. The propapain gene, obtained from Carica papaya fruit, is expressed in the yeast Saccharomyces cerevisiae. The gene was cloned into a FLAG epitope-tagging expression vector downstream of the yeast alpha mating factor (alpha-factor) secretion signal sequence. Expression of the heterologous propapain in yeast is controlled by the glucose-repressible alcohol dehydrogenase isoenzyme II promoter (ADH2). Glycosylated FLAG-tagged propapain is secreted by a so-called 'super secretor' strain, pmr1 (ssc1), into the culture supernatant where it accumulates to approximately 1.7 mg/l. The proregion contains three consensus N-linked glycosylation sites, whereas there are only two such sites in previously reported cDNA sequences. Removal of this third N-linked glycosylation site results in a drastic reduction in the level of protease activity present in the culture supernatant. Two different types of affinity chromatography were used to purify either propapain or papain. The propapain precursor is autoproteolytically activated to mature papain (M(r) = 24 kDa) using conditions reported previously. The kinetic parameters obtained agree well with the literature values. The yields of active papain are 10-fold higher than those previously reported for propapain in other yeast or bacterial expression systems. This, together with the ease with which mutant proteins can be made, makes this yeast advantageous for a structure-function analysis of recombinant wild-type and mutant papain, and possibly for other related cysteine proteases as well.

Cloning and characterization of theHansenula polymorpha homologue of theSaccharomyces cerevisiae PMR1 gene

“…Due to the properties of Pmr1p distinct from sarco/ endoplasmic reticulum and plasma membrane Ca 2+ pumps, it has been suggested that Pmr1p represents a new class of P-type Ca 2+ -ATPase that plays an important role in the secretory pathway (Sorin et al, 1997). Mutation in the PMR1 gene has also been shown to increase the secretion of some other heterologous proteins, and thus the pmr1 mutant strains have been considered as suitable hosts for the secretory production of various recombinant proteins that are poorly secreted by the wild type (Turner et al, 1991;Harmsen et al, 1993Harmsen et al, , 1996Ramjee et al, 1996).…”

A Novel High-Throughput Screen Reveals Yeast Genes That Increase Secretion of Heterologous Proteins

“…As a contrasting approach, the folding and secretion apparatus of yeast can be rationally tuned by overexpression or deletion of target genes thought to play a role in protein secretion. A nonexhaustive list of examples includes the overexpression of heavy chain binding protein (BiP), yielding increased production of single-chain antibodies (scFv) (35), and scTCR (33); overexpression of PDI, increasing yields of scFv (35), granulocyte colony-stimulating factor (44), and acid phosphatase (30); and deletion of the Golgi apparatus-resident calcium ATPase gene, PMR1, increasing the production yields of prochymosin (10) and propapain (28). However, this semirational approach requires preliminary knowledge of potential gene targets.…”

Heterologous expression of the plant cysteine protease bromelain and its inhibitor in Pichia pastoris

“…As protein yields also depend on cell density, production was upscaled to a 10 L bioreactor. Fermentation of P. -7), Ananain without N-and C-terminal propeptide (8)(9)(10)(11), Ananain with C-terminal propeptide (12)(13)(14)(15), Ananain with C-terminal propeptide (16)(17)(18)(19). As control supernatant of the wild type strain was used (1)(2)(3).…”