CHiC, a new tandem affinity tag for the protein purification toolbox

Abstract

In the present work we have constructed a new tandem affinity purification tag and used it to purify two different polypeptides, PcsB and ECL1 from Streptococcus pneumoniae. PcsB probably functions as a peptidoglycan hydrolase and is believed to be involved in splitting of the septum during cell division. ECL1 is the extracellular domain of the membrane spanning protein FtsX. Experimental evidence indicates that the ECL1 domain controls the activity of PcsB through direct interaction (Sham et al., 2011). The affinity tag consists of an N-terminal 6xHis-tag, a choline binding domain followed by a proteolytic site specific for the TEV (tobacco etch virus) endopeptidase. Based on the choline-binding His-tag combination the new 16.5kDa tag was named CHiC. CHiC-tagged PcsB and ECL1 were expressed in Escherichia coli and sequentially purified by employing diethylaminoethyl-cellulose affinity chromatography and Ni2+ immobilized metal affinity chromatography. After TEV digestion, the CHiC-tag, TEV-protease and undigested fusion protein were easily separated from the target protein in a single purification step. By using this method, 4–7mg of recombinant PcsB and ECL1 were obtained from one liter of cell culture with a purity estimated to be at least 95%. In addition, we found that the tag has the potential to function as a solubilisation partner as it markedly increased the solubility of PcsB. In sum, the CHiC-tag is a versatile tool that allows purification of milligram quantities of highly purified recombinant protein in only one or two steps.