Protein Expression and Purification

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 A600 » 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 CO chemical shifts measured using solid-state nuclear magnetic resonance spectroscopy. © 2008 Elsevier Inc. All rights reserved. ures been part es of ich­ia with rane dies. ium ium yield lim. with rcial fercentration, pH, and dissolved oxygen, the fermenter is expensive and not available in all laboratories. This paper describes the alternate but related approach of controlling growth parameters in shake flask fermentation. The culture was grown to maximum cell density in rich medium and then switched into fresh minimal medium prior to induction of protein expression [2]. Labeled amino acids were added to the minimal medium at induction and were incorporated into the expressed protein [3]. As stated earlier, one dif­fi­culty with membrane protein expression is the limited cell membrane space. Excess membrane protein is often stored as insoluble aggregates which are termed inclusion bodies. The amount of protein that can be recombinantly produced as inclusion bodies is up to 25% of the total cell mass, making inclusion bodies an attractive target for protein production [4]. However, the inclusion body protein has to be solubilized, purified, and properly folded in membranes or detergent. A typical protocol includes concomitant denaturation and solubilization using a solution containing a denaturant such as urea, purification, and then refolding with solutions containing detergent and possibly lipid vesicles [5–7]. In this paper, refolding was achieved by first mixing the solution of purified denatured fusion protein with a solurtani; While there have been a number of high-resolution struct of bacterial membrane proteins in recent years, there have fewer structures of viral and eukaryotic membrane proteins in because of dif­fi­culties with production of milligram quantiti pure and folded protein by heterologous expression in Esch­e­r coli [1]. This paper describes methods to address this problem a particular focus on production of isotopically labeled memb protein in E. coli for nuclear magnetic resonance (NMR) stu Isotopic labeling of proteins is usually done in a minimal med with consequent reduction in cell growth relative to rich med and lower production of protein. For membrane proteins, the may be reduced further because of higher hydrophobicity and ited cell membrane space needed to maintain native structure The protein production in this study was accomplished conventional shake flask fermentation rather than a comme fermenter. Although cell density can often be increased in a menter by controlling growth parameters such as carbon con * Corresponding author. Fax: +1 517 353 1793. E-mail addre­ss: [email protected] (D.P. Weliky). 1 Abbre­viat­ions use­d: NMR, nuclear magnetic resonance; LB, Luria–Be ll rights reserved. tion containing high concentration detergent and then dialyzing to reduce the denaturant concentration to a negligible level. The target “FHA2” influenza fusion protein in this study is prototypical for membrane enveloped viruses which initiate cellular -tetradecylphosphatidylchoG, isopropyl thiogalactoside; ouble-resonance; CD, circular J. Curt­is-Fisk e­t­ al. / Prot­e­in Expre­ssion and Purificat­ion 61 (2008) 212–219 213 infection by fusion between the viral and host cell membranes. The influenza virus is first endocytosed into the cell and fusion occurs between the viral and endosomal membranes at the endosomal pH of »5. The HA2 protein in the viral membrane catalyzes fusion and brings the two membranes together with consequent lipid mixing and formation of a fusion pore through which the viral nucleocapsid enters the cytoplasm [8]. HA2 has a 185 residue N-terminal “FHA2” ectodomain which lies outside the virus, a 25residue transmembrane region, and a short 10 residue C-terminal endodomain [9]. The N-terminus of HA2 and FHA2 is the »20 residue apolar “fusion peptide” region which is in the protein interior prior to endocytosis. After the pH in the endosome is reduced to »5, HA2 undergoes a large conformational change which exposes the fusion peptide. The fusion peptide then binds to the endosomal membrane and fusion ensues. High-resolution structures exist for the pre-fusion ectodomain complex crystallized from aqueous solution at pH 8 and for a “soluble ectodomain” of HA2 (residues 34–178) crystallized from aqueous solution at pH 4.4 [9,10]. In addition, there are liquid-state NMR structures of the fusion peptide in detergent micelles and electron spin resonance measurements of membrane location of the fusion peptide and of an HA2 ectodomain fragment representing residues 1–127 [11,12]. There have also been solid-state NMR measurements of local conformation at three residues in membrane-associated FHA2 whose sequence is displayed in Fig. 1 [13]. Functional relevance of FHA2 was demonstrated by induction of rapid vesicle fusion at pH 5.0 and much less fusion at pH 7.4 which correlated with the low pH of influenza virus/endosome fusion. In the earlier solid-state NMR study, the purified yield of the 23 kDa FHA2 protein expressed in E. coli was 3 mg/L culture. The present paper describes production of 20 mg/L quantities of isotopically labeled FHA2 which is a very good yield for a non-bacterial membrane protein expressed in E. coli [14,15]. Mate­ri­als and me­th­ods