Utilization of fermentation and purification strategies to enhance the yield of BmR1 antigen

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dc.contributor.authorNorsyahida Arifin
dc.date.accessioned2016-11-15T01:19:01Z
dc.date.available2016-11-15T01:19:01Z
dc.date.issued2010-03
dc.description.abstractBrugia Rapid ™ is an established diagnostic test that is commercially available to detect infection to both B. malayi and B. timori infections (brugian filariasis). The test is a dipstick lateral flow test format that utilizes BmRl recombinant antigen expressed by the clone Bm17DIIJlpPROEX™HTITOP 10. Due to a significant demand for the test in the market; there is a need to scale up the production of the recombinant antigen and increase the purification efficiency to reduce the production cost of the test. The cultivation of the recombinant bacteria was performed using fed-batch fermentation where cells were grown in a modified Terrific broth medium and glucose was fed exponentially at a controlled rate using Multifermenter Control Software (MFCS) for automated feeding during pre-induction and post-induction feeding. Varying the specific growth rate (11) prior to induction showed that a final cell concentration of 24.3 gIL was obtained at specific growth rate of 0.15 h-l with feeding at high rate during post-induction. However, further increase of the specific growth rate during pre-induction feeding does not produce a higher cell mass, in fact it decreased the BmRl recombinant protein production. It was found that the highest BmRl production of 9.82 gIL was obtained when the feeding was carried out at low constant rate (1.9 ml/min), combined with the specific growth rate controlled at 0.075 h-1 • It was also observed that at this feeding rate, the overall specific productivity, the fermentation yield coefficients [biomass yield (Y xis) and product yield (Y pIs)] was the highest amongst all the runs tested. This strategy has successfully controlled the accumulation of acetic acid by-inhibitory product below the reported growth inhibitory level of 1.26 gIL. The expression of BmRl recombinant antigen was found to be optimal with twice induction of 1 mM of IPTG concentration. Initiation of expression at mid log had generated significant amounts of the soluble protein. Therefore, the BmRl recombinant antigen was then purified under non-denaturing conditions using immobilized metal affinity chromatography. In order to increase the efficiency of purification process, various volumes of wash buffer, imidazole and salt concentration were performed. Imidazole at 20 mM was found to be the best concentration that gave the best yield of BmRl recombinant antigen while achieving sufficient purity. When 5 column volumes (CV) of washing buffer was used in the purification step, the production of BmRI recombinant antigen was 5.14 mg/l; as compared to 2.84 mg/l when 10 CV of washing buffer was employed. The overall production of the target protein was improved seven-fold compared to the conventional flask cultivation method.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/3070
dc.subjectBrugia Rapid is an established diagnostic test that is commercially availableen_US
dc.subjectto detect infection to both B. malayi and B. timori infectionsen_US
dc.titleUtilization of fermentation and purification strategies to enhance the yield of BmR1 antigenen_US
dc.typeThesisen_US
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