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

Abstract

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.

Simultaneous immunization of mice with Omp31 and TF provides protection against Brucella melitensis infection

“…Following purification over Ni-NTA agarose, the sizes of the expression products were verified by SDS-PAGE and Western blotting [13,36]. As expected, the molecular weight of recombinant rOmp31 and rTF corresponded to 28 and 59 kDa, respectively.…”

“…Contaminating endotoxins were eliminated during the purification step by adding 0.1% Triton X-114 to the washing buffer [37][38][39]. Purity of the recombinant proteins and their identity were assessed by SDS-PAGE, Coomassie blue staining, silver nitrate staining and Western blotting [36]. Briefly, purified recombinant proteins were size-separated by SDS-PAGE and the proteins transferred to a nitrocellulose membrane (BioRad, USA).…”

“…1 mM isopropyl-␤-d-thiogalactopyranoside (IPTG) was used to induce expression of the recombinant proteins. After induction, rTF was expressed as a soluble form and so could be purified under native conditions while rOmp31 was observed to be expressed as an insoluble form and required solubilization for purification [36]. Briefly, rOmp31 was solubilized in 8 M urea and at the next step re-folded in a stepwise dialyzing process with decreasing gradient of urea.…”

Smear grading and the Mantoux skin test can be used to predict sputum smear conversion in patients suffering from tuberculosis

“…In addition, biochemical and molecular tests were used to further confirm M. tuberculosis [ 15 ]. DNA typing, including RFLP, was used for final identification [ 16 ], [ 17 ], [ 18 ]. Susceptibility of isolates to isoniazid, rifampin, pyrazinamide, ethambutol, and streptomycin were determined as recommended by the CLSI (Clinical and Laboratory Standards Institute; formerly National Committee for Clinical Laboratory Standards – NCCLS) using the standard proportion method on a 7H10 agar medium.…”

Vaccines and Vaccine Candidates against Brucellosis

“…Brucella is a facultative intracellular pathogen causeing severe febrile illness in human, known as brucellosis (1). Brucellosis is the commonest zoonotic is the commonest zoonotic disease worldwide.…”