Histidines in affinity tags and surface clusters for immobilized metal-ion affinity chromatography of trimeric tumor necrosis factor alpha.
In order to achieve efficient IMAC (immobilized metal-ion affinity chromatography) purification of tumor necrosis factor alpha (TNF-alpha) and its analogs by a common chromatographic procedure, we tested four histidine-rich affinity tags attached to the N-termini of the trimeric TNF-alpha molecule. Using low cultivation temperature and appropriate protease deficient E. coli strains, 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 step. However, in comparison to model proteins bearing a surface histidine cluster, accumulation of the histidine-tagged proteins in E. coli was significantly reduced, even in protease deficient strains. In addition, the histidine tagged TNF-alpha proteins never displayed good chromatographic behavior, which was otherwise easily achieved with model proteins. Although the most 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 of the tag, with respect to its length, composition, and location. Histidines, relatively rigidly inserted in the structure, as in our model proteins, display superior chromatographic characteristics vis a vis flexible tags with the same total number of histidines.
Gaberc-Porekar
V
V
National Institute of Chemistry, Hajdrihova, Ljubljana, Slovenia.
Menart
V
V
Jevsevar
S
S
Vidensek
A
A
Stalc
A
A
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001704:Biopolymers; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry*; D006868:Hydrolysis; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D013499:Surface Properties; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000737:chemistry
D000345:Affinity Labels; D001704:Biopolymers; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D006639:Histidine