Isotopically labeled expression in E. coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein

Abstract

This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of E. 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 ~ 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 shift measured using solidstate nuclear magnetic resonance spectroscopy.

Native Conformation at Specific Residues in Recombinant Inclusion Body Protein in Whole Cells Determined with Solid-State NMR Spectroscopy

“…In addition, ~10 mg/L more FHA2 was purified after sonication of the aforementioned pellet in 8 M urea, and this denatured FHA2 could be refolded in detergent and reconstituted in membranes. 16 The relative band intensities in Figure 1a suggest that FHA2 is at least 10–20% of the total protein mass in the insoluble fraction and in unlysed whole cells and in the present study, the labeling/solid-state NMR method was applied to FHA2 in these two hydrated materials. 17, 18 The 13 CO NMR signals were dominated by FHA2 as evidenced by comparison of the “ S 0 ” (black) and “ S 1 ” (red) REDOR NMR spectra of the pellet for which the labeling targeted the Leu-98/Leu-99 unique sequential pair, Figure 1b.…”

“…However, growth only in minimal media provided <0.5 mg purified FHA2 per L culture and an alternate protocol was developed to first grow to high cell densities in rich media and to then switch to minimal media prior to expression. 12, 15, 16 Using this method, the purified yield of FHA2 from the soluble cell lysate was ~10 mg/L culture. This was likely the fraction of FHA2 which was incorporated in cell membranes because the lysis buffer contained N -lauroylsarcosine detergent which is effective at solubilizing membrane proteins.…”

HIV gp41 six-helix bundle constructs induce rapid vesicle fusion at pH 3.5 and little fusion at pH 7.0: understanding pH dependence of protein aggregation, membrane binding, and electrostatics, and implications for HIV-host cell fusion

“…2). Hairpin was expressed and purified as described previously (Curtis-Fisk et al 2008; Sackett et al 2009). Expression was done in BL-21 cells using the T7 expression system, and following bacteria growth, induction, and protein expression, the centrifuged cell pellet was lysed with glacial acetic acid.…”

Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines

“…Because of these complications, in vivo Escherichia coli expression produced low yields of only 2 mg/L, and the HA stem polypeptides accumulated as inclusion bodies (1). Although some refolding methods have been attempted (14)(15)(16), the recovery yields of soluble products have been low, and properly folded stable trimeric assembly has not been confirmed.…”