pPICHOLI vectors DNA

High-Efficient Protein Expression in Pichia pastoris & E. coli

• 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

Description
The 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) alcohol oxidase (AOX) 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.

The pPICHOLI vectors
The dual expression vectors pPICHOLI and pPICHOLI-HA 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 of 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.
Our pPICHOLI-HA vector includes a HA (hemagglutinin) epitope instead of the biotinylation sequence.

pPICHOLI-C carries the CUP1 promoter of Saccharomyces cerevisiae (instead of the AOX promoter) which has been shown to greatly reduce the induction time (11, 12). pPICHOLI-C has no T7-promoter and is not suited for prokaryotic protein expression.
Because of 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/µg DNA (6). Additionally, plasmids can be easily recovered from P. pastoris.

The pPICHOLI dual expression vectors include an RGS(His)6 tag as well as an additional in vivo biotinylation sequence (13) for sensitive detection and rapid purification of expressed proteins. Owing to the strong affinity of biotin to avidin, capture and screening assays are facilitated.

Kit Content

• pPICHOLI-1 vector DNA    10 µg
• pPICHOLI-2 vector DNA    10 µg
• pPICHOLI-3 vector DNA    10 µg
• pPICHOLI-C vector DNA    10 µg
• pPICHOLI-HA vector DNA    10 µg
• Sequencing primers    500 pmole each