Development of an Inducible Secretory Expression Vector and Host System for High Yield Production of Recombinant Protein
Research Square (Research Square)(2021)
Abstract
Abstract BackgroundEscherichia coli has been the most widely used recombinant protein expression system due to the availability of various protein expression vectors and ease of genetic manipulation. However, recombinant proteins expressed in E. coli are often contaminated with lipopolysaccharide (LPS) highly toxic to humans and must be removed from FDA-approved biologics, a process which requires extensive and expensive procedures. Gram-positive bacteria possess a single layer of cytoplasmic membrane free of LPS which make it ideal for producing recombinant protein. However, a lack of inducible protein expression systems limits a large-scale protein production in Gram-positive bacteria.ResultsThe HptARS is a three-component regulatory system in Staphylococcus aureus which senses extracellular glucose-6-phosphate and activates the uhpT gene promoter to facilitate uptake of extracellular G6P. To construct an inducible and secretory protein expression vector system, the promoter of the uhpT gene and the N-terminal signal peptide sequence of the hlb gene was fused in-frame with a C-terminal 6x-histidine sequence. For constitutive expression, we generated S. aureus expression host strain lacking the uhpT gene which could not uptake extracellular G6P, resulting in constitutive activation of HptARS system. With this newly established expression vector system and host strain, we demonstrated large-scale production of biologically active and highly pure staphylococcal leukotoxin E. ConclusionExtracellular expression of recombinant protein in LPS-free bacteria has a tremendous advantage in industrial production of FDA-approved biologics. Our newly established inducible and secretory expression vector system and S. aureus host strain will be useful to produce recombinant proteins for vaccine applications and other industrial purposes.
MoreTranslated text
Key words
inducible secretory expression vector,protein,high yield production
AI Read Science
Must-Reading Tree
Example
![](https://originalfileserver.aminer.cn/sys/aminer/pubs/mrt_preview.jpeg)
Generate MRT to find the research sequence of this paper
Chat Paper
Summary is being generated by the instructions you defined