Production of Brugia malayi BmSXP recombinant protein expressed in Escherichia coli

Abstract

A rapid antibody detection test is very useful for detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. One such kit, panLF Rapid TM (commercialized by Malaysian BioDiagnostic Research Sdn. Bhd.) had been developed in our laboratory for the detection of all species of filarial infections. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant Brugia malayi antigens, BmR1 and BmSXP. In this study, the growth of recombinant bacteria that produce BmSXP was optimized under shake flask fermentation for high yield of the recombinant antigen. The optimizations involved selection of suitable growth medium, IPTG concentration and induction time. The medium that yielded the highest biomass as well as total protein was Terrific Broth (TB) medium, which is an undefined medium. Initiation of induction of protein expression was found to be best at mid-log phase (OD600 = 1.5), with IPTG concentration of 1.0 mM, and harvest time at 9 h post-induction. This study showed that under the optimized conditions, the shake flask culture produced 4 g/L biomass (dry cell weight) of recombinant Escherichia coli BmSXP/pPROEXHTa/TOP10F', which yielded 2.42 mg/L of purified BmSXP recombinant antigen. The purified antigen was analyzed by SDS-PAGE and the antigenicity of protein was confirmed by Western blot.

Improved expression and immobilization of Geobacillus thermoleovorans CCR11 thermostable recombinant lipase

“…As large-scale production of organisms with extreme life conditions may be both difficult and expensive [3], the use of recombinant DNA techniques has made easier the production, purification, and characterization of several thermostable lipases [4,5]. Numerous studies have been conducted in order to improve recombinant heterologous gene expression using Escherichia coli as a host [1,[6][7][8][9]. Induction conditions for such systems are considered important parameters in the production of recombinant proteins, and they need to be carefully adjusted for every new expressed protein [10,11].…”

“…A thermoalkalophilic lipase from Geobacillus thermoleovorans CCR11, a thermophilic bacterium isolated from a hot spring in Veracruz, M éxico, has been previously cloned and expressed in E. coli using the pET-3b vector (Novagen), under the control of the inducible strong promoter T7 [4,12]. The recombinant protein LipMatCCR11 was the product of the expression system that contained the lipase structural gene minus the signal peptide, and exhibited a lipolytic activity (2.0 × 10 5 U/mg) that was 40-fold higher than the one produced by the native strain (5,500 U/mg); this recombinant lipase presented an optimal activity under a wide range of temperature (30-60 • C) and pH values (7)(8)(9)(10) [4]. In addition, when LipMatCCR11 was used in lyophilized form for the synthesis of flavors and fragrance esters in reactions of acidolysis, alcoholysis, and aminolysis with hexane as a reaction medium, it showed conversion percentages similar to those obtained with Candida antarctica lipase fraction B (CAL-B) proving to be a new and promising biocatalyst [13].…”