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Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115.

ABSTRACT: Human serum albumin (HSA) and human parathyroid hormone (1-34) [PTH (1-34)] fusion protein [HSA/PTH (1-34)] is a promising long-acting form of PTH (1-34) for osteoporosis treatment. Secretory expression of intact HSA/PTH (1-34) in Pichia pastoris GS115 was accompanied by two degradation fragments, with molecular weights around 66 kDa, in addition to the well-known ~45 kDa HSA-truncated fragment, resulting in a low yield of intact protein. In this study, two internal cleavage sites were identified in the PTH (1-34) portion of the fusion protein by Western Blot analysis. To minimize proteolytic cleavages, several protease genes including PEP4 (encoding proteinase A), PRB1 (proteinase B) and seven YPSs genes (yapsin family members) were knocked out respectively by disruption of the individual genes and the selective combinations. Reduced degradation was observed by single disruption of either PEP4 gene or YPS1 gene, and the lowest level of degradation was observed in a pep4△yps1△ double disruptant. After 72 h of induction, more than 80 % of the HSA/PTH (1-34) secreted by the pep4△yps1△ double disruptant remained intact, in comparison to only 30 % with the wild-type strain.

SUBMITTER: Wu M

PROVIDER: S-EPMC3656227 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Proteomic analysis of Pichia pastoris GS115

Project description:Pichia pastoris GS115 was cultured in YPD medium and protein were extracted and separated on SDS-PAGE followed by in-gel trypsin digestion. Protein were also digested in-solution using trypsin and fractionated using SCX and bRPLC. A total of 101 peptide fractions were analyzed on LTQ-Orbitrap Velos mass spectrometer. The raw data was searched using Mascot, Sequest and MS Amanda search algorithms against P. pastorisGS115 NCBI RefSeq database through Proteome Discoverer software suite.

2014-11-18 | PXD000627 | Pride

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

Project description: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.

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Enhancement of HSA-pFSHβ production by disrupting <i>YPS1</i> and supplementing N-acetyl-L-cysteine in <i>Pichia pastoris</i>.

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