title: Chemical cleavage of fusion proteins for high-level production of transmembrane peptides and protein domains containing conserved methionines.
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.
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Hu
authors.forename: Huajun
authors.initials: H
authors.lastname: Qin
authors.forename: Mukesh
authors.initials: M
authors.lastname: Sharma
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Cross
authors.forename: Fei Philip
authors.initials: FP
authors.lastname: Gao
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002210:Caprylates; D017124:Conserved Sequence; D005466:Fluorocarbons; D005982:Glutathione Transferase / Q000378:metabolism; D019811:Hydroxylamine / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002210:Caprylates; D005466:Fluorocarbons; D010455:Peptides; D011993:Recombinant Fusion Proteins; D019811:Hydroxylamine; C023036:perfluorooctanoic acid; D008715:Methionine; D005982:Glutathione Transferase