Construction and Characterization of a Medium Copy Number Expression Vector Carrying Auto-Inducible dps Promoter to Overproduce a Bacterial Superoxide Dismutase in Escherichia coli.

Abstract

Medium copy number expression vector and auto-inducible promoter could be a solution for producing recombinant therapeutic proteins in industrial scale regarding plasmid stability, cost, and product quality. This work aimed to construct a medium copy number pBR322-based expression vector carrying auto-inducible promoter, determine its ability to express heterologous gene, and study its segregational stability. Three stationary-phase promoters of Escherichia coli genes (gadA, dps and sbmC) were used to produce a superoxide dismutase from Staphylococcus equorum (rMnSODSeq) coding region from pBR322Δtet (pBR322-mini). Four plasmids were constructed with different promoters, i.e., T7 (pBMsod), gadA (pMCDsod), dps (pCADsod), and sbmC (pCDSsod) using pBR322-mini as backbone. Results showed that rMnSODSeq expression from pBMsod was significantly higher than that from pJExpress414sod (high copy number plasmid). Meanwhile, rMnSODSeq from pCADsod (auto-inducible promoter) was as high as from pBMsod (IPTG-inducible T7 promoter). rMnSODSeq expressed from pCADsod when bacterial cells entered stationary phase appeared as an active protein band of 23.5kDa when analyzed by zymography and SDS-PAGE. pCADsod displayed the highest stability compared with pBMsod and pJEXpress414sod by plasmid retention assay. We demonstrate the use of an auto-inducible dps promoter to express high level of heterologous protein, an SOD of S. equorum, from a stable expression vector with medium copy number.