Fig. 4.

Fig. 4. (A) The PCR product of the 1.1 kbp DNA fragment of the L-asparaginase gene of Burkholderia pseudomallei. The DNA fragment was analyzed on a 1.2% TAE agarose gel. Lane 1: DNA marker (Gel pilot wide range ladder 100 -Qiagen). Lane 2: 1.1 kbp DNA fragment PCR product of L-asparaginase gene. (B) Schematic diagram of the recombinant Burkholderia pseudomallei L-asparaginase overexpressions construct. The Lasparaginase gene was cloned downstream of the Tac promoter in the pGEX-2T DNA expression vector, which also contained the genes for lacI and lacZ repressors, pBR322 origin, and ampicillin resistance. (C) Induction time course for overexpression of L-asparaginase protein. Early to the mid-log culture of E. coli BL21 with Lasparaginase recombinant plasmid was induced at time 0 h with IPTG at a final concentration of 1 mM and samples were taken and analyzed by 10% SDS-PAGE gel at times indicated. Lane 2-8: protein marker, Lane 1: Sigma SD6H2 (MW 25,000-200,000 kDa). (D) The purification profile of the L-asparaginase protein on SDSPAGE. Lane 1: protein marker, Lane 2: E. coli L-asparaginase crude extract, Lane 3: Glutathione S sepharose 4B column-eluted L-asparaginase. (E) Western blot analysis with anti-GST antibody. Lane 1: crude extract, Lane 2: purified L-asparaginase.

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