Chemical cleavage of fusion proteins for high-level production of transmembrane peptides and protein domains containing conserved methionines |
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Authors: | Jian Hu Huajun Qin Timothy A. Cross Fei Philip Gao |
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Affiliation: | a Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, USA b National High Magnetic Field Laboratory, Tallahassee, Florida 32310, USA c Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, USA |
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Abstract: | Due to their high hydrophobicity, it is a challenge to obtain high yields of transmembrane peptides for structural and functional characterization. In the present work, a robust method is developed for the expression, purification and reconstitution of transmembrane peptides, especially for those containing conserved methionines. By using a truncated and mutated glutathione-S-transferase construct as the carrier protein and hydroxylamine (which specifically cleaves the peptide bond between Asn and Gly) as the cleavage reagent, 10 mg of the first transmembrane helix of CorA, a Mg2+ transporter from Mycobacterium tuberculosis, can be conveniently obtained with high purity from 1 L of M9 minimal media under optimized conditions. The biophysical properties of the peptide were studied by circular dichroism and nuclear magnetic resonance spectroscopy, and the results show that this CorA peptide is well folded in detergent micelles and the secondary structure is very similar to that in recent crystal structures. In addition, this CorA construct is oligomeric in perfluoro-octanoic acid micelles. The compatibility with the transmembrane peptides containing conserved methionines, the high yield and the simple process make the present method competitive with other commonly used methods to produce such peptides for structural and functional studies. |
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Keywords: | CD, circular dichroism CorA-TM1, the first transmembrane helix of CorA CSI, chemical shift index DPC, dodecyl phosphocholine ESI-TOF mass, electrospray ionization time-of-flight mass CNBr, cyanogen bromide GST, glutathione-S-transferase GuHCl, guanidine chloride HSQC, heteronuclear single quantum coherence IPTG, isopropyl-β- smallcaps" >d-thiogalactopyranoside LIC, ligation independent cloning MBP, maltose binding protein NMR, nuclear magnetic resonance PFO, perfluoro-octanoic acid TEV, tobacco etch virus protease TMPs, transmembrane peptides TGST, truncated glutathione-S-transferase SDS, sodium dodecyl sulfate |
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