OGS165

PSF-T7-NH2-OMPA SP-NCOI - BACTERIAL SECRETION PLASMID

plasmid vector for molecular cloning

• Synonyme(s) : cloning vector, expression vector, molecular cloning vector, plasmid, plasmid vector, snapfast vector, vector
• NACRES: NA.85
• UNSPSC Code: 12352200
• Promoter: Promoter name: T7; Promoter activity: inducible; Promoter type: phage
• Origin of replication: pUC (500 copies)
• Bacteria selection: kanamycin
• Reporter gene: none
• Peptide cleavage: no cleavage

Propriétés

• form: buffered aqueous solution
• mol wt: size 3900 bp
• bacteria selection: kanamycin
• origin of replication: pUC (500 copies)
• peptide cleavage: no cleavage
• peptide tag location: N-terminal
• promoter: Promoter name: T7; Promoter activity: inducible; Promoter type: phage
• reporter gene: none
• secretion signal: OmpA
• shipped in: ambient
• storage temp.: −20°C

Description

General description

This vector allows transcription of un-polyadenylated and un-capped RNA using the T7 bacteriophage polymerase. This vector also contains the Escherichia coli (E. coli) outer membrane protein A (OmpA) secretory signal peptide. When positioned at the N-terminus of a protein it should direct the protein to be secreted out of the bacterial cytosol and into the periplasmic space. The OmpA secretory tag is 21 amino acids in length. The signal peptide protein sequence is MKKTAIAIAVALAGFATVAQA. Cleavage occurs after the final Alanine residue. The peptide tag will be cleaved the mature protein during export. In this vector the signal peptide coding sequence has been positioned adjacent to the NcoI restriction site.

This technique can be used to increase di-sulphide bond formation and also reduces proteolytic degradation. This method also reduces contamination from endogenous proteins because very few E. coli proteins are secreted. A sub fraction of the protein secreted into the periplasmic space can also extracted from the growth media. It is still unclear why this occurs but it believed to reflect increased permeability of the outer membrane particularly during extended culture periods.

Secretion of proteins in E. coli can sometimes be difficult. Potential issues include protein toxicity within the periplasm variable secretion efficiency formation of inclusion bodies within the cytoplasm when using strong promoters incorrect dis-sulphide formation. Background to bacterial secretion systems including possible solutions to these problems can be found in Choi et al 2004 Applied Microbiology and Biotechnology 64 (625-635).

all our vectors (except those in the terminators category on our website) will contain a T7 terminator downstream of the multiple cloning site. This will enable transcription termination.

Promoter Expression Level:

Application

Cloning in a gene: This vector has been designed to allow the addition of a peptide tag to the end of a protein of interest using standard cloning techniques.Multiple Cloning Site Notes:

There is a start codon in the NcoI site can be removed by digestion with KpnI if required. The MCS for gene insertions extends from NotI to XbaI however the tag resides between the NotI and HindIII sites. There are Shine-Dalgarno sequences and KOZAK sequences aligned with the start codon of the peptide tag.

The ClaI to NheI sites have other functions such as adding C-terminal peptide tags second promoters or IRES expression components. The BsgI and BseRI restriction sites cleave within the stop codon in the XbaI site and allow the retrospective fusion of C-terminal peptide tags sequences if the stop codon is placed in this position.

Analysis Note

To view the Certificate of Analysis for this product, please visit www.oxgene.com

Other Notes

To view sequence information for this product, please visit the product page

Informations sur la sécurité

• Classe de stockage: 12 - Non Combustible Liquids
• Point d'éclair (°F): Not applicable
• Point d'éclair (°C): Not applicable