His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors

Abstract

We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His₆) tags in Escherichia coli by testing these on 20 human proteins. We looked at total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His₆ tags have a noticeable negative effect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with a C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins.

abbreviations:

BSA – bovine serum albumin; DBD – DNA binding domain; DCW – dry cell weight; EDTA – ethylenediaminetetraacetic acid; GFP – green fluorescent protein; IMAC – immobilized metal ion affinity chromatography; IPTG – isopropyl-β-d-thiogalactopyranoside; LB – Luria-Bertani; MES – 2-(N-morpholino) ethane sulfonic acid; OD – optical density; ORF – open reading frame; PCR – polymerase chain reaction; SDS-PAGE – sodium dodecyl sulfate polyacrylamide gel electrophoresis.