Cloning and expression analysis of synthetic MHETase gene under mutated pelB signal sequence in Escherichia coli BL21 (DE3).

Polyethylene terephthalate (PET) is widely used as raw material for clear, strong, and lightweight plastics that have been exploited in the packaging market. Although the use of plastics makes human's life easier, their waste has become a major environmental problem. Recycling plastics-based waste is still expensive and highly energy consuming. Recently two steps synergic degradation of PET has been discovered in Ideonella sakaiensis in which PET is converted into mono-(2-hydroxyethyl) terephthalic acid (MHET) catalyzed by PETase, and MHET is further hydrolyzed into terephthalic acid and ethylene glycol catalyzed by MHETase. This discovery offers a promising and eco-friendlier solution to the global environmental crisis of plastic pollution. In this work, we have cloned the MHETase gene synthetically designed based on IsMHETase. The synthetic gene was codon optimized for protein expression in Escherichia coli. Aiming to obtain extracellular MHETase, the recombinant enzyme was produced in E. coli BL21 (DE3) and its secretion was mediated by a mutated pelB signal sequence. Expression of MHETase was induced by IPTG at 37°C for 5 h. Intracellular, periplasmic, and extracellular fractions were extracted using different methods. Intracellular and periplasmic fraction was obtained from a lysate of sonicated and osmotic shocked cells, respectively, while extracellular fraction was yielded from concentrated cell free culture. Those three fractions were further analyzed by SDS-PAGE and Western Blot using anti-6X HisTag antibodies. We found that the recombinant MHETase was majority expressed as inclusion bodies and distinctively observed after purification of solubilized protein. Western Blot analysis of solubilized fraction showed a stronger signal at ~62 kDa than soluble intracellular protein fraction. Active MHETase were observed in refolded protein fraction and generated 0.4015 U.mL-1 of esterase activity. [ABSTRACT FROM AUTHOR]