Plasmid Info:

Plasmid Purpose:

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 SnapFast^TM 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.

Sequence and Map:

Other Info:

This plasmid contains three alternative transcription terminators for mammalian bacterial and bacteriophage (T7) expression. This means that only the promoter needs to be changed to alter the expression system you are using. We sell multiple promoters that can be used in each of these systems. The presence of each terminator does not reduce expression in the alternative systems.

Cloning:

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.

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.

IP Status:

This product is part of our SnapFast plasmid range, for more information on the Intellectual property status of this plasmid please click here