Reduced Proteolysis of Secreted Gelatin and Yps1-Mediated α-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 ␣-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 ␣-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 ␣-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.

Cloning and Characterization of the Glycoside Hydrolases That Remove Xylosyl Groups from 7-β-xylosyl-10-deacetyltaxol and Its Analogues

“…Theoretically, the matured peptide should have a molecular weight of 82 kDa. Further analysis revealed a sequence of Ile-Phe-Arg-Arg present in the C-terminal end of the first 35 amino acids, which is a typical Kex2 site ((Ali/ Arg)-Xaa-(Lys/Arg)-Arg2, where Ali and Xaa represent an aliphatic amino acid and an arbitrary amino acid, respectively) (41,42). The recombinant enzyme was mainly kept inside the recombinant yeast cells, even when the secreted expression vector pPIC9K was applied (Figs.…”

Degradation of HSA-AX15(R13K) when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast

“…It is well established that YPS1 is a glycophosphatidylinositiolanchored protein that localizes to cell membrane and partially to other different subcellular locations such as late Golgi apparatus and vacuole (Ash et al, 1995;Sievi et al, 2001). In addition, Werten and Wolf (2005) had cloned and characterized the YPS1 gene of P. pastoris. According to the above, it is supposed that the YPS1 gene-encoded product in P. pastoris may be responsible for the degradation of HSA-AX15(R13K).…”

Rational Design of a Novel Propeptide for Improving Active Production of Streptomyces griseus Trypsin in Pichia pastoris

“…1A). The four residues were inserted by digestion with restriction enzymes SnaBI and EcoRI and cleavage of the ␣-factor signal peptide by peptidases KEX2 and STE13 (12)(13)(14). However, no activity was detected when substituting the native propeptide with mammalian trypsinogen propeptide VDDDDK or its mutant VDDDDD (11).…”