Browsing by Author "Low, Kheng Oon"

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    Extracellular secretion of cyclodextrin glucanotransferase in escherichia coli by modification of hemolysin transport system
    (Universiti Teknologi Malaysia, 2011) Low, Kheng Oon
    Direct transport of recombinant protein from cytoplasm to the extracellular medium offers enormous advantages such as simple purification step and high specific activity. One of the approaches is through the hemolysin (Hly) transport system. The objective of this project is to construct an efficient extracellular protein secretion system using the Hly transport system by mutagenesis and process optimisation. Cyclodextrin glucanotransferase (CGTase) fused to a C-terminal 61 amino acid HlyA (HlyAs61) was shown to be secreted specifically by the Hly system, albeit at a low level (1.96 ± 0.21 U/ml). In order to make the system competitive for industrial application, protein engineering via error-prone PCR was performed on hly genes (hlyAs, hlyB and hlyD). Through screening of about 1 x 104 transformants generated by error-prone PCR, five mutants that showed a 35% to 217% increase in secretion level of CGT-HlyAs61 relative to the wild-type strain were isolated. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain. It was shown that mutant HlyB-V162A has the ability to secrete a wide range of recombinant proteins, as demonstrated on CGTase and cutinase. Combination of beneficial mutations further increased protein secretion in a range of 6 – 27% than mutant HlyB-V162A. In order to rationalize the effect of beneficial mutations, molecular models of HlyB were constructed using homology modeling. A putative substrate peptide binding pocket was proposed based on protein-protein docking between HlyB and HlyA signal sequence models. However, the effect of beneficial mutations towards increased-secretion phenotype was not observed. To further increase recombinant CGTase secretion level, a novel secretion system, termed as dual-plasmid system, was constructed in which gene expression for CGTase-HlyAs61 and Hly membrane proteins (hlyB and hlyD) were regulated by T7lac and PBAD promoter, respectively. At least 4-fold higher CGTase secretion level was obtained by the dual-plasmid system over the initial plasmid system. Subsequent optimisation via response surface methodology was carried out to maximise extracellular yield of recombinant CGTase. The optimised culture conditions were found to be 25.76 µM IPTG, 1.0% (w/v) arabinose and 34.7°C postinduction temperature which gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increment compared to the initial condition. An optimum expression of transported protein and translocation machineries (HlyBHlyD) is essential for maximum target protein secretion. Single-step purification of the extracellular CGTase-HlyAs61 by starch adsorption resulted in a near homogeneity protein sample. As a conclusion, novel mutants HlyB and plasmid system designs, which lead to increased protein secretion level, were constructed.