Cloning of SNAP-tag Fusions in pSNAP-tag(T7) (N9174)
Overview
Cloning by PCR
Although your protein of interest can be expressed with the SNAP-tag as either an N- or a C-Terminal fusion, we recommend cloning the gene of interest downstream of the SNAP26b gene in pT7-SNAP. The expression of the SNAP-tag at the N-Terminus may increase the expression level for weaker ex-pressing proteins.
Subclone your gene of interest into the pSNAP-tag(T7) Vector using the available restriction sites SbfI , AscI , BamHI which are located upstream of the stop codon, or one of these sites combined with EcoRI , EcoRV or XhoI. This will result in your protein being fused to the C-Terminus of the SNAP-tag. Include a stop codon where necessary.
Introduction
Primer Design and Cloning Hints:
- Design your PCR primers to include a sufficient overlap with the sequence of the gene you want to amplify.
- Adapt the flanking regions to the cloning destination in pT7-SNAP. If you add an upstream SbfI and a downstream BamHI site in the primers, cloning downstream of SNAP26b in pT7-SNAP should be straightforward.
- You may also want to include a stop codon at the C-Terminus of the fusion (in front of the downstream cloning site) in order to terminate translation at this position.
- In general, any linker peptide between the proteins should be kept short to avoid degradation by proteases. If required, specific protease cleavage sites can be introduced into the linker peptide.
- Care should be taken to design the cloning so that the fusion partners in the resulting construct are in frame.
- Perform the PCR reaction and subsequent cloning steps according to established protocols for molecular biology.
- After subcloning the gene of interest into pSNAP-tag(T7) as a fusion with the SNAP26b gene, the resulting plasmid can be used for expression of the SNAP-tag fusion proteins in a suitable E. coli host strain, such as T7 Express Iq (NEB #C3016) or T7 Express lysY/Iq (NEB #C3013). To subclone the gene of interest into pT7-SNAP fused to the N-Terminus of the SNAP-tag use the available restriction sites NdeI, KpnI and ApaI which are located upstream of the SNAP-tag.
Direct cloning can also be used to make fusions with the SNAP-tag. This is only possible if the fusion partner has compatible sites adjacent to the gene of interest. Although your protein of interest can be expressed with the SNAP-tag as either an N- or a C-Terminal fusion, we recommend cloning the gene of interest downstream of the SNAP26b gene in pSNAP-tag(T7). The expression of the SNAP-tag at the N-Terminus may increase the expression level for weaker expressing proteins.
The sequence of the SNAP26b gene is flanked by a number of restriction sites which can be found on the plasmid map. Suitable restriction sites provided for cloning downstream of SNAP26b include SbfI, AscI, BamHI which are located upstream of the stop codon, and EcoRI, EcoRV or XhoI downstream. This will result in your protein being fused to the C-Terminus of the SNAP-tag. The linker created by the SbfI/PstI site usually does not interfere with expression and activity of the SNAP-tag.
Care should be taken to design the cloning so that the fusion partners in the resulting construct are in frame.
After subcloning the gene of interest into pSNAP-tag(T7) as a fusion with the SNAP26b gene, the resulting plasmid can be used for expression of the SNAP-tag fusion protein in a suitable E. coli host strain.
Troubleshooting
If subcloning of the SNAP-tag with your gene of interest does not work, reconfirm all the cloning steps (primer design, choice of restriction site, etc.) If all steps are confirmed as being correct, then try the cloning using different restriction sites. Be sure to include a positive control for the ligation reaction.
Alternatively try to subclone the SNAP-tag gene into an expression vector already containing your gene of interest.
