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DNA Vector Systems

pPICHOLI Shuttle Vector System

High-efficient 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 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

Vector Maps of pPICHOLI

pPICHIA-1/-2/-3 and -HA: ColE1 ori: ColE1 origin of replication; AOX promotor: alcohole dehydrogenase promotor; Zeocin: zeocin resistance; PARS1: P. pastoris autonomous replicating sequence; RGSHis(6): Poly(His)-Tag; BS: biotinylation sequence;

mcs of pPICHOLI

Sequence data of the cloning site of pPICHOLI-1. pPICHOLI-2 is lacking one of the G bases (dark shaded box) in front of the Sal I restriction site, pPICHOLI-3 is lacking both G bases upstream Sal I. The ATG start and the TAA stop codons are marked in bold and italic. *pPICHOLI-HA includes the 6xHis tag in front of the BamH I site and a HA tag (instead of the biotinylation sequence) behind the Bam HI site.
Note: Bam HI is not a singular restriction site!

ColE1 ori: ColE1 origin; Zeocin: zeocin resistance; PARS1: P. pastoris autonomous replicating sequence; RGSHis(6): Poly(His)-Tag; BS: biotinylation sequence;
Note: Bam HI is not a singular restriction site! pPICHOLI-C has no T7-Promotor!

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 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.

The new 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 reduce the induction time greatly (11, 12). pPICHOLI-C has no T7-promotor and is not suited for procaryotic protein expression.

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/µg DNA (6). Additionally, plasmids can be easily recovered from P. pastoris. The pPICHOLI dual expression vectors include a RGS(His)6 tag as well as an additional in vivo biotinylation sequence (13) for sensitive detection and rapid purification of expressed proteins. Due to the strong affinity of biotin to avidin, capture and screening assays are facilitated.

ORDER INFORMATION, SHIPPING & STORAGE:
order#	description	amount*
PPICH	pPICHOLI-1 vector DNA pPICHOLI-2 vector DNA pPICHOLI-3 vector DNA pPICHOLI-C vector DNA pPICHOLI-HA vector DNA sequencing primers	10 µg 10 µg 10 µg 10 µg 10 µg 500 pmole each
Shipped at room temperature (RT). Lyophilized plasmid DNA can be stored at 4°C. Once the DNA has been dissolved in sterile water or buffer we recommend storage at -20°C.

Please download: handbook

pPICHOLI sequences, restrction sites marked (pdf, 20kb)

single sequences:
pPICHOLI-1 , pPICHOLI-1, pPICHOLI-3 , pPICHOLI-HA , pPICHOLI-C
(txt, 4kb each)

For further Vector System products please download the Vector Systems brochure (PDF, 3,9 MB)

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