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Why is my bacterial vector not expressing my recombinant protein?

The plasmid is placed in an inappropriate host strain

The plasmid may not be expressed in an inappropriate host strain for induction. Most vectors from VectorBuilder are shipped as E. coli stock in the cloning host strain Stbl3 (this information is also indicated on the vector report). Stbl3 is the preferred cloning host due to its ability to maintain the stability of the plasmid, but it may not be suitable for recombinant protein expression. For example, for pET, the IPTG-induced recombinant protein expression requires the T7 RNA polymerase to be expressed in the host strain, which is not present in Stbl3. As such, bacterial expression vectors typically require transferring the plasmid into an appropriate host strain such as BL21(DE3) for proper induction.

The protein expressed on the vector is “problematic”

Sometimes when the protein being expressed is insoluble, misfolded, improperly cleaved, or toxic to the bacterial host, there may appear to be poor induction of the recombinant protein. In this case, you will need to optimize your induction system (see below), or express your gene of interest in a more tolerant host strain or from an alternative expression vector.

Your induction system is not optimized

Depending on the gene to be expressed, the expression vector and the host strain, you may need to consider optimizing the following things in your induction system: OD600 (usually between 0.6 and 0.8); concentration of the inducing agent (e.g. IPTG or L-arabinose) cannot be too low or too high; duration of induction cannot be too short or too long; induction temperature needs to be optimized especially when you are dealing with “problematic” proteins (see above), and different temperatures can be tested (e.g. 16°C, 25°C, 30°C, 37°C, etc.). In rare cases, for unclear reasons, different clones of the same expression vector may show different induction behavior, so you may need to pick a number of single colonies to test individually and select the one that has best induction performance.

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