Cloning strategy, production and purification of proteins with exopeptidase-cleavable His-tags

Abstract

Here, we present a cloning strategy for the production of recombinant proteins tagged with a polyhistidine sequence that can be cleaved by the exopeptidase, DAPase. The method can be used with most commonly available vectors and results in the expression of a His-tag protein that can be purified in its native form regardless of its natural sequence. This approach takes advantage of the TAGZyme system for the removal of amino-terminal affinity tags. Tag removal is accomplished either with DAPase (a recombinant dipeptidyl peptidase) alone or in combination with two accessory enzymes, Qcyclase and pGAPase. The system has been used for the production of intracellular proteins in Escherichia coli and can be applied to other expression hosts for the production of secreted proteins or proteins that require post-translational modification. The production of human interleukin 1β in E. coli is used as an example to illustrate this method. The complete protocol from initial PCR to the production of a detagged protein with its authentic N terminus can be performed within 5 days.

NOTE: In the version of this article initially published online, the text for Step 21 of the procedure was incorrect. It should read: “Prepare an overnight culture (1.8 l) and centrifuge in a Sorvall for 20 min at 10,000g, 4 °C.” This error has been corrected in all versions of the article.