Dataset Information

Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2 disruptant.

ABSTRACT: Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae alpha-factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the alpha-factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the alpha-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.

SUBMITTER: Werten MW

PROVIDER: S-EPMC1087524 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

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Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2 disruptant.

Werten Marc W T MW de Wolf Frits A FA

Applied and environmental microbiology 20050501 5

Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae alpha-factor preproleader used to direct high-le ...[more]

PMID: 15870316

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Project description:In the methylotrophic yeast Pichia pastoris, we expressed the rat acetylcholinesterase H and T subunits (AChEH and AChET respectively), as well as truncated subunits from rat (W553stop or AChETDelta, from which most of the T-peptide was removed) and from Bungarus (V536stop, or AChENAT, or AChEDelta, reduced to the catalytic domain). We show that AChEH and AChET subunits are processed into the same molecular forms as in vivo or in transfected mammalian cells, but that lytic processes converting amphiphilic forms into non-amphiphilic derivatives appear to be more active in yeast. The production of glycophosphatidylinositol (GPI)-anchored molecules (dimers, with a small proportion of monomers) demonstrates that P. pastoris can correctly process a mammalian C-terminal GPI-addition signal. Truncated rat and Bungarus AChE molecules, which exclusively generated non-amphiphilic monomers, were released more efficiently and thus produced more AChE activity. In the hope of increasing the production of AChE, we replaced the endogenous signal peptide by yeast prepeptides, with or without a propeptide. We found that the presence of a propeptide, which does not exist in AChE, does not prevent the proper folding of the enzyme, and that it may either increase or decrease the yield of secreted AChE, depending on the signal peptide. Surprisingly, the highest yield was obtained with the endogenous signal peptide. For all combinations, the yield was 2-3 times higher for Bungarus than for rat AChE, probably reflecting differences in the folding efficiency or stability of the polypeptides. The Michaelis constant (Km), the constant of inhibition by excess substrate (Kss) and the catalytic constant (kcat) values of the recombinant AChEs obtained both in P. pastoris and in COS cells, were essentially identical with those of the corresponding natural enzymes, and the Ki values of active-site and peripheral-site inhibitors (edrophonium, decamethonium, propidium) were similar.

Dataset Information

Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2 disruptant.

Publications

Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2 disruptant.

Similar Datasets

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