pTrcHis TOPO® TA Expression Kit From Invitrogen

Purified recombinant proteins can be an extremely valuable commodity for any laboratory. Purified protein can be used as a substrate for enzyme assays, as positive controls for Western blots and ELISAs and even as immunogen for production of polyclonal antibodies. However, the supply of commercially available proteins is often limited, making it impossible to find a source of your protein of interest. In addition, recombinant protein can be cripplingly expensive with a few micrograms costing hundreds of dollars.

If your goal is to simply have a positive control for an ELISA assay, there is little need for an extensively tested protein that has been demonstrated to have biological activity. However, you will in the end often be paying for just this kind of validation, even though it is not necessary for your application. So what alternative is there for obtaining your samples while maintaining a reasonable balance between cost and lab time?

Traditional cloning and expression techniques often required extensive amounts of time developing and optimizing protocols tailored to each individual target. Today, the availability of pre-packaged kits of every shape and variety has removed much of the burden of optimizing protocols from the end user. Enhancements such as TA cloning and the use of PCR to screen colonies has cut huge amounts of time, often days, from the time required to get from cDNA to established clone.

Invitrogen’s pTrcHis TOPO® TA Expression Kit provides all the necessary reagents to go from cDNA to recombinant protein. The kit comes in two packages, one for storage at –80ºC that contains 50 µl aliquots of chemically competent TOP10 E. coli, S.O.C. medium, and a supercoiled control plasmid. The second box, which is stored at –20ºC contains 200 ng of linearized, topoisomerase I-activated pTrcHis-TOPO® vector, sterile water, dNTPs, 10X PCR buffer, a control template and primers, primers for sequencing or PCR screening, and an expression control plasmid. The only reagents necessary for the user to supply are Taq polymerase, the cDNA template and specific primers to amplify the target gene.

The cloning strategy takes advantage of the fact that Taq polymerase adds a single overhanging 3’ adenosine residue, to clone any PCR product between the two overhanging 5’ thymidines at each end of the cloning site. The PCR products are introduced into the vector using a topoisomerase reaction that only requires 5 minutes to run to completion, saving significant time over traditional ligation strategies. Transformation is preformed immediately following the topoisomerase reaction. This allows the entire PCR amplification, ligation and transformation to be done in a single day. By screening the resulting colonies by PCR it is possible to identify and inoculate candidate expression clones in two days.

In my evaluation of this kit, I have been quite pleased with its ease of use and the speed with which one can go from PCR reaction to pilot expression. I have successfully cloned and expressed inserts ranging in size from ~300 bp to >1.5kb. The only notable drawbacks of the kit that I have encountered have been the relatively small number of transformants and the expense of the kit’s initial purchase. In addition it appears that the LacI^q/LacO repressor is somewhat leaky, making it difficult to clone proteins that exhibit toxic effects on the bacteria. In reality though, these are minor points when you consider that a single positive clone is all that is required for successful production of recombinant protein and that this kit really is quite complete, even including competent cells for transformation.