Pichia pastoris expression vector with methanol inducible promoter AOX1 and the alpha factor secretion peptide. Also has a histidine synthesis selection marker.
Product Name: pSF-AOX1-A-factor-SnapFusion-HIS4
Product Code: OG3582
Size (bp): 7353 bp
Bacterial Antibiotic Selection: KanR
Origin and Compatibility: pUC high copy derived from pBR322
Bacterial Copy Number: 500-700 per cell
This vector is designed for the expression of a gene of interest (GOI) in the methylotrophic yeast, Pichia pastoris. Once the GOI has been cloned into the MCS, the plasmid is then linearised and transformed into an appropriate P. pastoris strain by electroporation. The expression construct becomes integrated into the genome. Expression of the GOI is induced by addition of methanol to the culture medium.This vector enables secretion of your GOI using the upstream alpha-factor secretion signal.This vector contains the SnapFusion MCS for the easy insertion of peptide tags.Promoter Expression Level:
This promoter is inducible by methanol and exhibits high expression under these conditions. In the absence of methanol the promoter is tightly regulated and shows only basal levels of expression.About the Peptide Tag:
This plasmid contains an full length Alpha Factor (A-FactorFL) secretory peptide to allow proteins to be exported from the cytosol. Alpha factor is an endogenous yeast protein that is naturally secreted into the supernatant. The wild-type Alpha Factor protein contains a series of repeating protein sequences that are cleaved out by cellular proteases that recognise a specific site. This produces a series of small peptides that contain the mature alpha peptide sequence. This plasmid contain the wild-type signal peptide from the full length Alpha factor gene but also the coding sequence that leads up to the first endogenous protease cleavage site. The addition of these sequences has been demonstrated to provide more consistent secretion of proteins in comparison to using just the signal peptide alone. All of the wild-type protein sequences will be removed during protein maturation and export by endogenous proteases.
This plasmid contains the poly-adenylation signal from the Alcohol Oxidase 1 Promoter (AOX1) gene from Pichia pastoris to terminate transcription.
To insert your gene:
1: Amplify your gene with primers designed using this spreadsheet
2: Cut the plasmid with either BseRI or BsgI.*
3: Cut your gene with the enzyme you added using the spreadsheet (any of AcuI BpmI BpuEI BseRI BsgI EciI).
4: Clone the gene into the plasmid using DNA ligase.
Using this method with an N-terminal tag plasmid will result in the tag coding sequence immediately followed by your genes ATG start codon at the join. This results in a seamless fusion of the two sequences with no extra bases being added. Using this method on C-terminal tag plasmids will convert your genes stop codon into a TAC (Tyr Y) codon followed by the plasmid tag coding sequence. This results in no extra bases between your gene and the tag. See the diagram below for more information.
*Please note that insect expression plasmids cannot be cut with BsgI only BseRI because of unavoidable conflicting sites in the backbone. Also Yeast plasmids cannot be cut with BseRI because of unavoidable restriction sites in the backbone.
Using this technique will create a gene fragment that can be ligated into any or our >1500 peptide and reporter tag plasmids. If you use one of the other techniques below (Gibson InFusion Seamless or LIC) you will need new primers for every vector you clone into because the arms of homology will change according to the tag plasmid you are cloning into.
If you find that your gene sequence has sites in it that make using this cloning strategy difficult you can still use one of the alternative methods below (e.g. standard cloning or Gibson cloning).
Open the Primer Design Tool to help you design primers for cloning your gene in our SnapFusion technique.2: Standard Enzymes:
If you are not concerned about leaving a few extra bases between the tag coding sequence and your gene you can clone your gene into the vector using standard cloning restriction enzymes. This strategy will require you to choose which enzymes you want to use to clone your gene.
Open the Primer Design Tool which provides primers with different enzyme choices positioning your gene as close to the tag as possible in each case. Please note that standard enzymes will always leave additional nucleotides between your gene and the tag but using the spreadsheet will ensure the tag and gene are in frame.3: Gibson cloning/InfusionHD/GeneArt Seamless/Ligase Independent Cloning (LIC) Methods:
These cloning techniques use reagents sold by other companies and allow you to fuse sequences together using enzymes that chew back the DNA to leave overlapping ends/overhangs. The subsequent method of joining the DNA depends on the kit used. To use one of these techniques you can either design your own primers or you can use the spreadsheet below to help with the design.
Open the Primer Design Tool to help you design primers for cloning your gene using Gibson assembly InfusionHD GeneArt Seamless cloning or Ligase Independent Cloning (LIC) techniques.
This product is part of our SnapFast™ plasmid range, for more information on the intellectual property status of this plasmid and the terms of our licences please click here.