Purification of native proteins from the cytoplasm and periplasm of Escherichia coli using IMAC and histidine tails: A comparison of proteins and protocols

Abstract

Several proteins were purified in a single step from Escherichia coli in native form using fused histidine tails and immobilized metal affinity chromatography (IMAC). The procedures reported and compared are those for antibody fragments (single-chain Fv fragments and V L domains) carrying a C-terminal (His) 5 tail purified from the periplasm, the E. coli disulfide isomerase carrying a C-terminal (His) 6 tail purified from the periplasm, and yeast citrate synthase carrying both a N-terminal and a C-terminal (His) 5 tail purified from the cytoplasm. Because of blocks in the periplasmic folding process, the investigated murine single-chain T-cell receptor could be purified only in denatured form, despite being soluble, thereby demonstrating the sensitivity of the native purification to the folding state. A comparison of columns and metal ions showed that Zn 2+-iminodiacetic acid and Ni 2+-nitrilotriacetic acid gave equally good one-step purifications of V L domains and Fv fragments, but only in this combination of metal and chelator. The buffer is of secondary importance, and the preferred method of elution depends on the stability of the protein. In a crystal structure determination of the V L domain carrying the (metal-free) histidine tail, no significant electron density beyond the first two histidine residues was detected, implying a disordered structure of the unliganded affinity tail. When IMAC is used under denaturing conditions, an E. coli protease can be co-purified and refolded or co-purified under native conditions, and sensitive proteins can be digested during their in vitro refolding. Plasmid-encoded wild-type RTEM β-lactamase was obtained as a side product in pure form by IMAC in a single step.