Attachment of Histidine Tags to Recombinant Tumor Necrosis Factor-Alpha Drastically Changes Its Properties

Abstract

When studying two different histidine tags attached to the N-termini of the trimeric cytokine tumor necrosis factor alpha (TNF), the biological activity — measured as cytotoxicity on the L-929 cell line — of both tagged proteins was drastically reduced. The longer His10 tag reduced cytotoxicity to approximately 16% and the shorter His7 tag to 6% of the activity of their nontagged counterparts. After removal of the tags, biological activities reverted to the expected normal values, which clearly shows the key role of the attached histidine tags in diminishing biological activity. Studies on the mechanism of these effects revealed no specific interactions and showed that even the natural flexible N-terminus of TNF presents a steric hindrance for receptor binding, while any extension of the N-terminus increases this hindrance and consequently reduces biological activity. Also, in other proteins, the ligand or substrate binding sites may be hindered by histidine tags, leading to wrong conclusions about biological activity or other properties of the proteins. Thus caution is advised when using His-tagged proteins directly in screening procedures or in research.

Changed loading conditions and lysate composition improve the purity of tagged recombinant proteins with tacn‐based IMAC adsorbents

“…The ability to completely remove these new tags was also a major factor in their design, a process often required for the production of recombinant proteins intended for structure-function studies or use in therapeutic applications [28][29][30][31][32].…”

hUNG2 Is the Major Repair Enzyme for Removal of Uracil from U:A Matches, U:G Mismatches, and U in Single-stranded DNA, with hSMUG1 as a Broad Specificity Backup

“…The degradation problem was partially overcome by expressing the protein containing an N-terminal His tag. For functional analysis we decided to avoid histidine tags, however, because this may seriously affect the functional properties of some proteins (31). In the present system, the highest relative amount of fulllength hUNG2 at the time of cell harvest was observed using E. coli BL21 (ompT Ϫ , lon Ϫ ), in which ϳ50% of the protein appeared unprocessed.…”

Effect of His6-tagging of anterior gradient 2 protein on its electro-oxidation

“…Introduction of an affinity tag contributed to improvement in the biochemical properties of the target recombinant protein, such as improved protein yield [4], prevention of proteolysis [5], increased solubility [5], etc. On the other hand, adding tag can change protein conformation [6], inhibit enzyme activity [7], alter biological activity [8] etc., but to our knowledge, no information about the effect of His-tagging on the protein interactions with electrically charged surfaces was reported. In recent years, numerous fusion tags have been developed for recombinant protein production [1][2][3].…”