As with many other laboratories, we rely heavily on large-scale recombinant protein expression in our effort to conduct genetic and proteomic analysis of parasite-host cell interactions. There are several different protein expression systems to consider, but we find bacterial expression to be the easiest and least costly. However, high expression of heterologous proteins in bacteria usually results in the production of insoluble, incorrectly folded proteins that are commonly found in inclusion bodies. Another common problem is degradation of the product by bacterial proteases. Such events are clearly protein specific, with overall size and complexity playing a major role in determining how well the recombinant foreign proteins are expressed. Lowering the temperature, optimizing inducer agent concentration or utilizing N-terminal fusion proteins that promote solubility have been used to combat this common problem. One plausible reason for improper folding and inclusion body formation is that over-expression of foreign proteins overcomes the ability of bacterial chaperones to fold the proteins properly.
An alternative approach would be to over-express bacterial molecular chaperones (e.g. dnaK, dnaJ, groEL, groES, grpE, and trigger factor) in order to aid in protein folding. The Takara Chaperone Plasmid Set consists of a “chaperone team” of five different types of plasmids. Each plasmid is designed to express several chaperones in combination that can then work cooperatively to fold the foreign protein. The plasmids are first used to transform your standard bacterial competent cell host (e.g. E. coli BL21 (DE3) or JM109 cells). After isolating positive transformants, the cells are made chemically competent again. Note that these plasmids are chloramphenicol resistant allowing the use of a second antibiotic as a selecting agent (e.g. ColE1-type plasmids containing an ampicillin resistance gene marker). The cells are retransformed with your expression plasmid containing your gene of interest to produce a co-expression system. Co-transformed cells are selected on plates carrying chloramphenicol and ampicillin. In our case, we normally use a pBAD expression vector (ampR) which expresses the foreign protein under the control of the araB promoter. Conveniently, the Takara plasmids have either the araB and/or Pzt-1 (tet) promoter controlling the expression of the molecular chaperone set for the specific plasmid. In the case of the latter, a tetR gene is also present in the plasmid, allowing the unaffected growth of E. coli in the presence of 5 ng/ml or less of tetracycline. In our system, expression of both the chaperone team and our gene of interest are induced by the addition of L-arabinose (2-20%).
This is an amazingly straightforward system, allowing a laboratory to generate several aliquots of pre-transformed competent cell lines in order to determine the most suitable chaperone team to play with. However, depending on the size of your expression plasmid, the number of co-transformants can be very, very low (and this should be expected). Nevertheless, for the expression of foreign fusion proteins below 30 kD (enough for antibody development), this is not a problem! In the end, you only need a couple of positive co-transformants to move forward. We did several side-by-side comparisons of singly transformed cells (pBAD vector) with co-transformants. We found a marked difference in solubility for our expressed protein, wherein the recombinant protein was found entirely in the soluble fraction following standard cell lysis of the co-transformed cells! The singly transformed cells performed as expected with ~55-65% of the recombinant protein remaining in the cell pellet (despite several repeated extractions). As with everything, optimization is still required since different chaperone teams can have different effects on the expression of the protein.
Chaperone Plasmid Set From Takara Mirus Bio
Ease of use and confirmed, enhanced soluble expression of recombinant proteins.
A little time-consuming to make the primary transformed cells but once done, there really is not much of any drawback in trying this product.
The Bottom Line
Very highly recommended.