Utilization of fermentation and purification strategies to enhance the yield of BmR1antigen
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Date
2010
Authors
Arifin, Norsyahida
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Abstract
Brugia Rapid TM 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 Bml7DJII/pPROEX™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 usmg 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 (!l) prior to induction showed that a final
cell concentration of 24.3 g/L was obtained at specific growth rate of 0.15 h-1 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 g/L 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 (Ypts)] 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 BmR1
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
fcund 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 BmRl recombinant antigen
was 5.14 mg/1; as compared to 2.84 mg/1 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.
The sensitivity and specificity of the purified BmRl recombinant antigen produced in
this study was confirmed by Western blot and two other immunoassays techniques
namely ELISA and immunochromatographic rapid test. By the ELISA technique, it
was found that the BmRl recombinant antigen produced in this study retained its
Description
Keywords
Fermentation , BmR1antigen