An effective extracellular protein secretion by an ABC transporter system in <Emphasis Type="Italic">Escherichia coli</Emphasis>: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production |
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Authors: | Kheng Oon Low Nor Muhammad Mahadi Raha Abdul Rahim Amir Rabu Farah Diba Abu Bakar Abdul Munir Abdul Murad Rosli Md Illias |
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Institution: | (1) Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia;(2) Malaysia Genome Institute, Ministry of Science, Technology and Innovation, UKM-MTDC Smart Technology Center, 43600 Bangi, Selangor, Malaysia;(3) Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43600 Selangor, Malaysia;(4) School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Selangor, Malaysia; |
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Abstract: | Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific
activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette)
transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design
and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular
recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving
six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant
CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted
extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml,
resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of
about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient
protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage. |
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