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PPICH - pPICHOLI Shuttle Vector System

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High-efficient dual protein expression in Pichia pastoris & E.coli

The novel pPICHOLI vectors have been designed for heterologous gene expression in the yeast P. pastoris as well as in the prokaryote E. coli. The vectors contain an inducible (yeast) alcohole dehydrogenase (AOX) promoter (pPICHOLI-C:CUP1 promoter) and an E. coli T7 promoter as well as sequences allowing autonomous replication both in P. pastoris and E. coli. Thus, vector linearization is no longer required and small amounts of DNA are sufficient to successfully transform P. pastoris. The integrated PARS sequence enables simple recovery of plasmids from yeast. Time-consuming subcloning into a number of expression vectors including testing for a successful gene expression is no longer necessary. A multiple cloning site enables convenient ligation of DNA fragments into the vectors.

FEATURES:

  • the prokaryotic expression system is simple to handle and allows a cost-effective and high-level production of heterologous proteins
  • P. pastoris/pPICHOLI system is a powerful eukaryotic expression system showing rapid growth at high densities combined with the strong AOX or CUP1 promoter, respectively
  • ideally suited for expression of soluble proteins with post-translational modifications and those (eukaryotic) proteins causing problems when expressed in E. coli (e.g. proteins toxic to E. coli)
  • easy cloning and high transformation efficiencies
  • convenient affinity purification and detection of recombinant proteins


THE pPICHOLI VECTORS:

The dual expression vectors pPICHOLI and pPICHOLI-C combine eukaryotic and prokaryotic promoter elements. Phage T7 promoter, including the ribosomal binding site of the major capsid protein, promotes the efficient bacterial expression and is placed downstream from the P. pastoris promoter. The strong alcohol oxidase promoter (AOX) is tightly regulated, since protein expression is completely repressed when grown on glucose and maximally induced when grown on methanol. pPICHOLI is available with a multiple cloning site in three different reading frames to simplify cloning in frame with the tags (pPICHOLI-1, pPICHOLI-2, pPICHOLI-3). pICHOLI-1 (3579 bp) has two G bases directly upstream of the Sal I site. pPICHOLI-2 (3578) and pPICHOLI-3 (3577 bp), respectively, are lacking one or both of these G bases.
pPICHOLI-C carries the CUP1 promoter of Saccharomyces cerevisiae (instead of the AOX promoter) which has been shown to reduce the induction time greatly. Due to the use of a common selection marker zeocin, the sizes of the shuttle vectors remain small (~3.6 and 3.2 kb, respectively), hence they remain convenient for handling, cloning and transformation. By integration of a Pichia specific autonomous replicating sequence (PARS1) into the pPICHOLI vectors, linearization is no longer required and the transformation efficiency is increased up to 105 transformants/Āµg DNA. Additionally, plasmids can be easily recovered from P. pastoris. The pPICHOLI dual expression vectors include a double tag consisting of an RGS(His)6 and a HA (hemagglutinin) epitope as well as an additional in vivo biotinylation sequence for sensitive detection and rapid purification of expressed proteins. Due to the strong affinity of biotin to avidin, capture and screening assays are facilitated.

PACKAGING: pPICHOLI (1-3) vector DNA (5ug ea), pPICHOLI-C vector DNA (5ug), sequencing primers