Abstract
The limited amount of fusion protein transported into cytosol milieu has made it challenging to obtain a sufficient amount for further applications. To avoid the laborious and expensive task, T7 promoter-driving pET-30a(+) coding for chimeric gene of thymidine phosphorylase and core streptavidin as a model system was constructed and transformed into a variety of E. coli strains with T7 expression system. Our results demonstrated that the pET-30a(+)-TP-coreSA/Lemo21(DE3) system is able to provide efficient expression of soluble TP-coreSA fusion protein for purification. Moreover, the eluted TP-coreSA fusion protein tethered on biotinylated A549 carcinoma cells could effectively eliminate these malignant cells after administrating prodrug 5′-DFUR.
Highlights
The bacterium Escherichia coli is the most convenient and widely used system for expression of recombinant proteins [1]
CoreSA has enhanced biotin-binding features compared to full-length SA because the removal of non-functional terminal regions promotes the binding with less steric hindrance [7]
Some chimeric proteins such as scFv and CD47 fused with core streptavidin (coreSA) affinity tag have been reported to be produced in E. coli [8,9,10,11]
Summary
The bacterium Escherichia coli is the most convenient and widely used system for expression of recombinant proteins [1]. The E. coli strains with the presence of T7 promoter-based plasmids have been extensively employed to express proteins. CoreSA has enhanced biotin-binding features compared to full-length SA because the removal of non-functional terminal regions promotes the binding with less steric hindrance [7]. Some chimeric proteins such as scFv and CD47 fused with coreSA affinity tag have been reported to be produced in E. coli [8,9,10,11]. Our results showed that Lemo21(DE3), transformed with the pET-30a(+) coding for TP-coreSA chimeric gene, is able to express substantial amount of soluble TP-coreSA fusion protein for time-saving and cost-effective purification. The functionality of the purified TP-coreSA fusion protein was confirmed by its effectiveness of killing biotinylated A549 lung adenocarcinoma cells tethered with TP-coreSA via biotin-SA binding and treated with prodrug 5 -DFUR
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