title: Effects of IMAC specific peptide tags on the stability of recombinant green fluorescent protein.
abstract: 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 His6 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.
authors.affiliation: ARC Special Research Centre for Green Chemistry, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Widakowich
authors.forename: Chunfang
authors.initials: C
authors.lastname: Zhang
authors.forename: Simon
authors.initials: S
authors.lastname: Harris
authors.forename: Khosse
authors.initials: K
authors.lastname: Mitri
authors.forename: Glenn
authors.initials: G
authors.lastname: Powers
authors.forename: Kirk-Ski
authors.initials: KS
authors.lastname: Troung
authors.forename: Milton T W
authors.initials: MT
authors.lastname: Hearn
mesh_terms: D002846:Chromatography, Affinity; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry*; D017510:Protein Folding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins