Isotopically labeled expression in E. coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein.
This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of Escherichia coli shake flask culture. This yield is sufficient for NMR structural studies and the protein production methods are simple, straightforward, and rapid and likely applicable to other recombinant membrane proteins expressed in E. coli. The target FHA2 protein is the full ectodomain construct of the influenza virus hemagglutinin protein which catalyzes fusion between the viral and the cellular endosomal membranes during infection. The high yield of FHA2 was achieved by: (1) initial growth in rich medium to A(600) approximately 8 followed by a switch to minimal medium and induction of protein expression; and (2) obtaining protein both from purification of the detergent-soluble lysate and from solubilization, purification, and refolding of inclusion bodies. The high cell density was achieved after optimization of pH, oxygenation, and carbon source and concentration, and the refolding protocol was optimized using circular dichroism spectroscopy. For a single residue of membrane-associated FHA2 that was obtained from purification and refolding of inclusion bodies, native conformation was verified by the (13)CO chemical shifts measured using solid-state nuclear magnetic resonance spectroscopy.
Curtis-Fisk
Jaime
J
Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
Spencer
Ryan M
RM
Weliky
David P
DP
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002942:Circular Dichroism / Q000379:methods; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods / Q000592:standards; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012015:Reference Standards; D012995:Solubility; D013997:Time Factors; D014760:Viral Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D002247:Carbon Isotopes; D014760:Viral Fusion Proteins; D005947:Glucose; D005990:Glycerol