Effects of IMAC specific peptide tags on the stability of recombinant green fluorescent protein

Gabriel Widakowich, Chunfang Zhang, Simon Harris, Khosse Mitri, Glenn Powers, Kirk-Ski Troung, Milton Thomas William Hearn

Research output: Contribution to journalArticleResearchpeer-review

12 Citations (Scopus)


Immobilized metal ion affinity chromatography (IMAC) using peptide affinity tags has become a popular tool for protein purification. An important feature dictating the use of a specific affinity tag is whether its structure influences the properties of the target protein to which it is attached. In this work we have studied the influence on protein stability of two novel peptide affinity tags, namely NT1A and HIT2, and compared their effect to the commonly used hexa-histidine tag, all attached to the C-terminus of a enhanced green fluorescent protein (eGFP). A comparison of the influence of C- or N-terminal orientation of the tags was also carried out by studying the NT1A tag attached at either terminus of the eGFP. Protein stability was studied utilising guanidine hydrochloride equilibrium unfolding procedures and CD and fluorescence spectroscopy. The novel peptide affinity tags, NT1A and HIT2, and the His(6) tag were found to not affect the stability of eGFP. Although these results are protein specific, they highlight, nevertheless, the need to employ suitable characterisation tools if the impact of a specific peptide tag on the folded status or stability of a recombinant tagged protein, purified by immobilized metal ion affinity chromatographic methods, are to be rigorously evaluated and the appropriate choice of peptide tag made. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 1048-1053, 2011
Original languageEnglish
Pages (from-to)1048 - 1053
Number of pages6
JournalBiotechnology Progress
Issue number4
Publication statusPublished - 2011

Cite this