title: Membrane transport piece by piece: production of transmembrane peptides for structural and functional studies.
abstract: Membrane proteins are involved in all cellular processes from signaling cascades to nutrient uptake and waste disposal. Because of these essential functions, many membrane proteins are recognized as important, yet elusive, clinical targets. Recent advances in structural biology have answered many questions about how membrane proteins function, yet one of the major bottlenecks remains the ability to obtain sufficient quantities of pure and homogeneous protein. This is particularly true for human membrane proteins, where novel expression strategies and structural techniques are needed to better characterize their function and therapeutic potential. One way to approach this challenge is to determine the structure of smaller pieces of membrane proteins that can be assembled into models of the complete protein. This unit describes the rationale for working with single or multiple transmembrane segments and provides a description of strategies and methods to express and purify them for structural and functional studies using a maltose binding protein (MBP) fusion. The bulk of the unit outlines a detailed methodology and justification for producing these peptides under native-like conditions.
authors: Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors: Grant
authors: G
authors: Kemp
authors: Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors: Larry
authors: L
authors: Fliegel
authors: Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.|National Institute for Nanotechnology, University of Alberta, Edmonton, Alberta, Canada.
authors: Howard S
authors: HS
authors: Young
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D011994:Recombinant Proteins
keywords: bacterial expression; hydrophobic peptides; maltose binding protein; membrane proteins; molecular structure; organic extraction