Single step protocol to purify recombinant proteins with low endotoxin contents

Rationale: New vaccine approaches are needed for Pseudomonas aeruginosa, which continues to be a major cause of serious pulmonary infections. Although Th17 cells can protect against gram-negative pathogens at mucosal surfaces, including the lung, the bacterial proteins recognized by Th17 cells are largely unknown and could be potential new vaccine candidates. Objectives: We describe a strategy to identify Th17-stimulating protein antigens of Pseudomonas aeruginosa to assess their efficacy as vaccines against pneumonia.Methods: Using a library of in vitro transcribed and translated P. aeruginosa proteins, we screened for Th17-stimulating antigens by coculturing the library proteins with splenocytes from mice immunized with a live-attenuated P. aeruginosa vaccine that is protective via Th17-based immunity. We measured antibody and Th17 responses after intranasal immunization of mice with the purified proteins mixed with the Th17 adjuvant curdlan, and we tested the protective efficacy of vaccination in a murine model of acute pneumonia. Measurements and Main Results: The proteins PopB, FpvA, FptA, OprL, and PilQ elicited strong IL-17 secretion in the screen, and purified versions of PopB, FpvA, and OprL stimulated high IL-17 production from immune splenocytes. Immunization with PopB, which is a highly conserved component of the type III secretion system and a known virulence factor, elicited Th17 responses and also enhanced clearance of P. aeruginosa from the lung and spleen after challenge. PopB-immunized mice were protected from lethal pneumonia in an antibody-independent, IL-17-dependent manner. Conclusions: Screening for Th17-stimulating protein antigens identified PopB as a novel and promising vaccine candidate for P. aeruginosa.Keywords: vaccine; pneumonia; Th17; IL-17; Pseudomonas aeruginosa Pseudomonas aeruginosa is a major cause of serious, often antibiotic-resistant, lung infections in humans, particularly in patients receiving mechanical ventilation and those with cystic fibrosis (1, 2). Most P. aeruginosa vaccines developed to date, including those based on the LPS O antigen (3), the outer membrane proteins F and I (4, 5), or the type III secretion system component PcrV (6), have relied on conventional protective mechanisms-namely, antibody-mediated opsonophagocytic killing and/or antibody-mediated toxin inhibition. Although LPS O antigen-based vaccines can mediate high levels of immunity to P. aeruginosa, the protection is limited to strains having the same LPS serogroup (3). The failure of the Federal Hyperimmune Immunoglobulin Trial (7), which found no benefit in critically ill adults passively immunized with P. aeruginosa LPS O antigenspecific IgG, perhaps best illustrates that antibody-mediated protective mechanisms are not sufficient.Th17 cells have recently been shown to mediate antibodyindependent host defense against Klebsiella pneumoniae (8), although the bacterial proteins recognized by the Th17 cells in those studies were not fully characterized. In our own evaluations of live-attenuated P. ...

show abstract

“…The His-tagged proteins were purified using a previously described method (20) with slight modifications detailed in the online supplement.…”

Section: Expression and Purification Of Proteins From E Colimentioning

confidence: 99%

Th17-stimulating Protein Vaccines Confer Protection against Pseudomonas aeruginosa Pneumonia

Jin

Duan

et al. 2012

Am J Respir Crit Care Med

115

157

View full text Add to dashboard Cite

show abstract

“…The recombinant protein was purified by affinity chromatography using GSTresin (GenScript, Nanjing, China). To remove the majority of contaminating endotoxins, we washed the column thoroughly with cold PBS containing 0.1% Triton X-114 (Sigma-Aldrich, St. Louis, MO) and then PBS at 4˚C (20). The proteins were finally eluted by a buffer containing 10 mM glutathione (SigmaAldrich) and 50 mM Tris-HCl (pH 7.5), dialyzed in PBS three times and adjusted to the concentration of 1 mg/ml.…”

Section: Recombinant Protein and Antiserum Productionmentioning

confidence: 99%

Collaboration between a Soluble C-Type Lectin and Calreticulin Facilitates White Spot Syndrome Virus Infection in Shrimp

Wang

et al. 2014

The Journal of Immunology

View full text Add to dashboard Cite

White spot syndrome virus (WSSV) mainly infects crustaceans through the digestive tract. Whether C-type lectins (CLs), which are important receptors for many viruses, participate in WSSV infection in the shrimp stomach remains unknown. In this study, we orally infected kuruma shrimp Marsupenaeus japonicus to model the natural transmission of WSSV and identified a CL (designated as M. japonicus stomach virus–associated CL [MjsvCL]) that was significantly induced by virus infection in the stomach. Knockdown of MjsvCL expression by RNA interference suppressed the virus replication, whereas exogenous MjsvCL enhanced it. Further analysis by GST pull-down and coimmunoprecipitation showed that MjsvCL could bind to viral protein 28, the most abundant and functionally relevant envelope protein of WSSV. Furthermore, cell-surface calreticulin was identified as a receptor of MjsvCL, and the interaction between these proteins was a determinant for the viral infection–promoting activity of MjsvCL. The MjsvCL–calreticulin pathway facilitated virus entry likely in a cholesterol-dependent manner. This study provides insights into a mechanism by which soluble CLs capture and present virions to the cell-surface receptor to facilitate viral infection.

show abstract

“…Endotoxins are lipopolysaccharides (LPS) associated with the outer membrane of Gram-negative bacteria. The inherent toxicity of these LPSs has a profound effect on biochemical events of certain cells and can interfere with in vitro experiments (6).The threshold level of endotoxin for intravenous applications is set to 5 endotoxin units (EU) per kg body weight and hour by all pharmacopoeias (7). Because of this toxicity, the removal of even minute amounts is essential for safe administration or biological cell-based application.…”

Section: Introductionmentioning

confidence: 99%

Optimization and Efficient Purification in Production of Brucella melitensis Recombinant HSP A and TF Proteins With Low Endotoxin Contents

Ghasemi

Salari

Pourmand

et al. 2013

Jundishapur J Microbiol

View full text Add to dashboard Cite

Background:The development of an effective subunit vaccine against brucellosis is a research area of intense. But optimization of recombinant proteins production in Escherichia coli and content of endotoxins associated with final recombinant proteins are very important. Objectives: In the present study, expression and purification of Brucella melitensis rHSP and rTF were optimized to reduce endotoxin contaminants. Materials and Methods: pDEST-tf and pDEST-hsp were transformed into E. coli BL21 (DE3), and then B. melitensis recombinant HSPA and TF proteins were overexpressed. Purification of these proteins was optimized to remove most of endotoxin contaminants from the end product using 0.1% Triton X-114 in washing buffers. Results: An endotoxin reduction of less than 0.05 EUmg/1 was achieved with protein recovery close to an 80% yield. Conclusions: As this new protocol requires only one step to simultaneously purify tagged proteins and eliminate endotoxins, it represents a substantial advantage in time, effort, and expense.

show abstract

Single step protocol to purify recombinant proteins with low endotoxin contents

Cited by 144 publications

References 26 publications

Th17-stimulating Protein Vaccines Confer Protection against Pseudomonas aeruginosa Pneumonia

Th17-stimulating Protein Vaccines Confer Protection against Pseudomonas aeruginosa Pneumonia

Collaboration between a Soluble C-Type Lectin and Calreticulin Facilitates White Spot Syndrome Virus Infection in Shrimp

Optimization and Efficient Purification in Production of Brucella melitensis Recombinant HSP A and TF Proteins With Low Endotoxin Contents

Contact Info

Product

Resources

About