Histidines in affinity tags and surface clusters for immobilized metal-ionaffinity chromatography of trimeric tumor necrosis factor alpha

In order to achieve efficient IMAC (immobilized metal-ion affinity chromato graphy) purification of tumor necrosis factor a (TNF-alpha) and its analogs by a common chromatographic procedure, we tested four histidine-rich affin ity tags attached to the N-termini of the trimeric ThTF-alpha molecule. Usi ng low cultivation temperature and appropriate protease deficient E. coli s trains, it was possible to obtain intact, full-length proteins with NHis2Xa and HisArg tags, which could be purified to over 95% purity in a single st ep. However, in comparison to model proteins bearing a surface histidine cl uster, accumulation of the histidine-tagged proteins in E. coli was signifi cantly reduced, even in protease deficient strains. In addition, the histid ine tagged TNF-alpha proteins never displayed good chromatographic behavior , which was otherwise easily achieved with model proteins. Although the mos t popular hexa-histidine tag is generally recognized as very convenient for single step isolation of monomeric proteins, our results with trimeric TNF -alpha indicate that oligomeric proteins may require further optimization o f the tag, with respect to its length, composition, and location. Histidine s, relatively rigidly inserted in the structure, as in our model proteins, display superior chromatographic characteristics vis a vis flexible tags wi th the same total number of histidines. (C) 1999 Elsevier Science B.V. All rights reserved.