Protein Expression and Purification Core FacilityCloning
The classical way of cloning genes into different expression vectors is by using restriction enzymes. We have adopted a strategy of parallel cloning and expression into a variety of vectors containing the same multiple cloning sides. The gene of interest is cloned in parallel into a variety of expression vectors containing different tags and/or fusion partners, and into vectors for a variety of expression systems. This approach enables us to screen multiple constructs in parallel to find the best one for further scale-up of expression and purification.
Although this is a well-established and widely used system in our lab as well other labs, there are alternative techniques introduced here in more detail that can be used for cloning a larger number of target genes when restriction enzyme-based cloning becomes more difficult. The presence of different restriction sites makes it necessary to find individual combinations of suitable enzymes increasing effort and costs by using different enzymes with possibly different incubation conditions.
The cloning strategy consists of the following steps:
- the gene of interest is amplified by PCR.
- the PCR product is cloned into a specific cloning or expression vector using one of the cloning methods described below.
- the sequence of a positive clone is checked by sequence analysis.
- the gene of interest is sub-cloned into a variety of expression vectors (for different expression systems) using a fixed set of restriction enzymes or specific recombination sites.
For expression in E.coli, we have expression vectors with the following tags and fusion partners:
|N-terminal His6-tag||ZZ-tag (ZZ-tag)||
|N-terminal His6-tag||thioredoxin (TrxA)||
|N-terminal His6-tag||glutathione-S-transferase (GST)||
|N-terminal His6-tag||maltose binding protein (MBP)||
|N-terminal His6-tag||disulfide oxidoreductase (DsbA)||
|N-terminal His6-tag||disulfide isomerase (DsbC)||
|N-terminal His6-tag||SUMO1 (*)||
|N-terminal His6-tag||SUMO3 (*)||
(*) Due to specific sequence requirements, the SUMO-tag vectors do not contain the same arrangement of cloning sites of the other vectors of our pETM-series.
You can find more detailed information on our SUMO-fusion vectors.