Optimization of recombinant expression of polyethylene terephthalate-degrading enzyme from Ideonella sakaiensis in Escherichia coli BL21(DE3)
2023
Jo, E.J. | Jo, B.H.
A polyethylene terephthalate (PET) degrading enzyme from Ideonella sakaiensis (IsPETase) is a promising enzyme for the green disposal and valorization of PET waste due to its high activity at relatively lower temperatures. However, the low- level soluble expression of IsPETase in Escherichia coli BL21 (DE3), the most widely used host for recombinant protein production, hampers the large-scale application of IsPETase. Recently, an improved soluble expression of IsPETase was demonstrated by using NEXT tag, an intrinsically disordered solubility enhancer. In this study, two strategies were attempted to further enhance the soluble expression of both IsPETase and NEXT-IsPETase: the optimization of culture conditions and the chaperone co-expression. The soluble expression of both enzymes was slightly improved under optimal conditions compared to the standard 37℃ and 1 mM IPTG conditions, although the culture condition was not the primary factor for controlling the soluble expression of the enzymes. When the chaperones (GroEL/GroES or DnaK/DnaJ/GrpE) were co- expressed, the soluble expression was only trivially improved (for GroEL/GroES) or even decreased (for DnaK/DnaJ/GrpE). In addition, the chaperone DnaJ was co-purified along with the enzymes, which can complicate the downstream process. In conclusion, the expression of NEXT-fused IsPETase without chaperones under optimal conditions would be the best option for maximizing enzyme production in E. coli BL21(DE3).
显示更多 [+] 显示较少 [-]