Cloning and expression of polyethylene terephthalate hydrolase (PETase) gene under mutated pelB signal peptide in Eschericia coli BL21 (DE3).

Waste is a big global issue that has a substantial influence on the environment and human health. One type of waste that is notoriously difficult to decompose and requires a considerable amount of time to degrade is polyethylene terephthalate (PET) plastic. Due to its remarkably persistent properties, the accumulation of PET waste poses a major threat to the ecosystem. Several attempts have been made to address the problem of PET plastic waste, one of which is to use an enzymatic approach to accelerate the decomposition of PET. PET hydrolase (PETase), found in Ideonella sakaiensis, is a potential agent for the biodegradation of PET. In this study, we used a synthetic biological approach (synthetic DNA) to develop a PETase biocatalyst, in which the PETase gene was chemically synthesized, cloned into a pET-22b vector, and then expressed in E. coli BL21 (DE3) with IPTG induction. During the construction process, the mutated N-terminal pelB signal sequence was included with the expectation that the end-product PETase could be either secreted into periplasmic or excreted to the culture medium. Several different extraction methods were utilized, including intracellular (cytoplasmic), periplasmic, and extracellular extraction. In addition, Western Blot Analysis was carried out to confirm that the target protein had been expressed. The pET22b(+)-petase was verified using restriction enzymes, and the result showed that the gene was properly cloned. The SDS-PAGE and western blot result showed that the pET22b-IsPETase has been successfully expressed and secreted, but the amount is still low so that further optimization needs to be done. [ABSTRACT FROM AUTHOR]