Optimizing synthesis and expression of transmembrane peptides and proteins.
Structural studies of full-length membrane proteins have been hindered by their hydrophobicity and low expression in a variety of systems. However, a simplifying aspect of membrane protein folding is that individual transmembrane segments or membrane protein fragments have been observed to represent independent folding domains, and as such, can facilitate the study of packing interactions between TM helices, and the collection of structural information regarding membrane proteins. This review focuses on two categories of techniques--total peptide synthesis and bacterial expression--that can each be optimized for preparation of transmembrane protein segments. First, synthesis of hydrophobic transmembrane peptides that are N- and/or C-tagged with solubilizing residues such as lysine can improve manipulation of the transmembrane core in a variety of biophysical experiments. In this context, we describe general protocol considerations during the synthesis, cleavage, and purification stages of these peptides to identify appropriate parameters that combine to improve yields of hydrophobic peptides. Second, bacterial expression of membrane protein fragments is a useful tool for producing large quantities of hydrophobic protein segments. Targeting protein expression within Escherichia coli can facilitate purification, while attaching the hydrophobic construct to a hydrophilic fusion protein can amplify expression. We show that adapting protein constructs to comply with expression host specifications, in concert with thorough exploration of expression conditions such as the type of media used for expression, temperature, and cell strain, can significantly improve protein yields.
Cunningham
Fiona
F
Division of Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Department of Biochemistry, University of Toronto, Toronto, Ont., Canada.
Deber
Charles M
CM
D000595:Amino Acid Sequence; D004926:Escherichia coli; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary
D008565:Membrane Proteins; D010446:Peptide Fragments