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. 2014 Oct:102:1-7.
doi: 10.1016/j.pep.2014.07.005. Epub 2014 Jul 19.

A scalable method for biochemical purification of Salmonella flagellin

Raphael Simon  1 , Brittany Curtis  2 , Vehid Deumic  3 , Jennifer Nicki  3 , Sharon M Tennant  2 , Marcela F Pasetti  4 , Andrew Lees  5 , Philip W Wills  3 , Marco Chacon  3 , Myron M Levine  6
Affiliations

A scalable method for biochemical purification of Salmonella flagellin

Raphael Simon et al. Protein Expr Purif. 2014 Oct.

Abstract

Flagellins are the main structural proteins of bacterial flagella and potent stimulators of innate and adaptive immunity in mammals. The flagellins of Salmonella are virulence factors and protective antigens, and form the basis of promising vaccines. Despite broad interest in flagellins as antigens and adjuvants in vaccine formulations, there have been few advances towards the development of scalable and economical purification methods for these proteins. We report here a simple and robust strategy to purify flagellin monomers from the supernatants of liquid growth culture. Phase 1 flagellins from Salmonella enterica serovars Typhimurium (i epitope) and Enteritidis (g,m epitopes) were purified directly from conditioned fermentation growth media using sequential cation- and anion-exchange chromatography coupled with a final tangential flow-filtration step. Conventional porous chromatography resin was markedly less efficient than membrane chromatography for flagellin purification. Recovery after each process step was robust, with endotoxin, nucleic acid and residual host-cell protein effectively removed. The final yield was 200-300 mg/L fermentation culture supernatant, with ∼45-50% overall recovery. A final pH 2 treatment step was instituted to ensure uniformity of flagellin in the monomeric form. Flagellins purified by this method were recognized by monoclonal anti-flagellin antibodies and maintained capacity to activate Toll-like Receptor 5. The process described is simple, readily scalable, uses standard bioprocess methods, and requires only a few steps to obtain highly purified material.

Keywords: Flagellin; Purification; Salmonella; Scalable; TLR5; Vaccine.

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Figures

Fig. 1
Fig. 1
Fermentation growth and accumulation of flagellin in culture supernatants of Salmonella Enteritidis reagent strain CVD 1943. Kinetics of growth and FliC accumulation in CVD 1943 fermentation culture were monitored by optical density at 600 nm (A) and SDS–PAGE with Coomassie staining (B).
Fig. 2
Fig. 2
Binding, wash, and elution of secreted FliC in CVD 1943 fermentation supernatants by cation-exchange membrane chromatography. (A) Chromatogram with absorbance at 280 nm (solid line) and 254 nm (dashed line); (B) SDS–PAGE with Coomassie stain of (M) Molecular weight standards, (1) fermentation culture supernatant, (2) diluted and pH adjusted fermentation supernatant, (3) column loading flow-through, (4) peak absorbance fraction from wash 1, (5) peak absorbance elution fraction. All lanes are from the same gel and exposure.
Fig. 3
Fig. 3
Anion-exchange membrane chromatography on cation-exchange membrane eluates of CVD 1943 secreted flagellin. (A) Chromatogram of A280 nm (solid black line) and A254 nm (dashed line) with indicated treatment steps denoted; (B) SDS–PAGE with Coomassie stain for: (1) anion exchange starting material, (2) flow through fraction, (3) 150 mM NaCl eluate, (4) 300 mM NaCl eluate. Molecular weight marker is denoted (M).
Fig. 4
Fig. 4
Concentration and monomerization of flagellins after anion-exchange chromatography. (A) CVD 1943 and (B) CVD 1925: (left panel) in-process material was analyzed by SDS–PAGE with Coomassie staining: (M) Molecular weight standards, (1) 150 mM NaCl anion exchange membrane eluate [10 μg], (2) 30 kDa TFF permeate, (3) 30 kDa TFF concentrated retentate [25 μg], (4) 30 kDa TFF concentrated-diafiltered retentate [25 μg], (5) post-pH 2 incubation [25 μg], (6) 0.2 μm filtrate [20 μg]; (right panel) SEC-HPLC of 0.2 μm filtered purified FliC measuring absorbance at 280 nm.
Fig. 5
Fig. 5
Reactivity of final purified flagellin proteins with anti-flagellin antibodies assessed by Western blot and ELISA. (A) Post 0.2 μm filtration final purified samples were loaded at 0.25 μg/well, separated by SDS–PAGE and transferred to nitrocellulose membranes for detection with a pan flagellin antibody: (1) CVD 1943 FliC, (2) CVD 1925 FliC; (B) CVD 1943 FliC coated onto ELISA wells, reacted with different monoclonal antibodies against common (CB7IH2, AE9IB4) or serovar specific (JB11IG4, CA6IE2) epitopes of S. Enteritidis FliC, tested in multiple concentrations; results represent absorbance values from replicate wells.
Fig. 6
Fig. 6
Functional innate immune bioactivity of S. Enteritidis CVD 1943 and S. Typhimurium CVD 1925 flagellins purified from fermentation culture supernatants. HEK293 cells stably transformed with a firefly luciferase reporter gene under control of NF-κB were seeded in 96-well plates and treated with media alone or the indicated concentration of CVD 1925 or CVD 1943 FliC. Luciferase levels were determined, and are presented as the average and standard error from duplicate wells.
Supplementary Fig. 1
Supplementary Fig. 1
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