Expression and refolding of recombinant human alpha-tocopherol transfer protein capable of specific alpha-tocopherol binding.
alpha-Tocopherol transfer protein (alpha-TTP) is a cytosolic protein found predominantly in mammalian liver that is proposed to be responsible for the stereoselective uptake of alpha-tocopherol from the diet. Although recombinant alpha-TTP has been reported previously, little detail has been provided about the yields and competency of the recovered protein at binding tocopherols and other ligands. In this work, we report the successful expression and refolding of a recombinant human alpha-TTP. Ligation-independent cloning generated a construct in pET-30 encoding an alpha-TTP fusion protein (pET-30/ttp) containing a six-histidine tag and an S-tag, each cleavable by a separate protease upon expression in Escherichia coli. Overexpression of the protein led to the formation of inclusion bodies that were solubilized in 8 M urea and purified by metal chelate affinity chromatography. Another construct in pET-28b (pET-28b/ttp) provided a soluble protein product after expression that contained a 40-amino-acid N-terminal extension, which can be reduced to 21 amino acids by cleavage with thrombin. The success of different refolding experiments was assessed using a Lipidex gel-based tocopherol binding assay. The best recovery of refolded recombinant alpha-TTP fusion capable of binding alpha-tocopherol was provided by matrix-assisted refolding in the presence of 0.5 M arginine. Cleavage of the fusion protein with Factor Xa successfully generated the full-length wild-type protein with no additional N-terminal amino acids. The resulting purification scheme provides recombinant alpha-TTP in good yield and purity for investigation of both its structure and its binding affinities for different ligands including natural and synthetic tocols.
Panagabko
Candace
C
Department of Chemistry and Centre for Biotechnology, Brock University, St. Catharines, Ontario, Canada.
Morley
Samantha
S
Neely
Steve
S
Lei
Huangshu
H
Manor
Danny
D
Atkinson
Jeffrey
J
D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D024502:alpha-Tocopherol / Q000378:metabolism*
D002352:Carrier Proteins; D008024:Ligands; D011994:Recombinant Proteins; C017763:alpha-tocopherol transfer protein; D024502:alpha-Tocopherol