Engineering the flagellar type III secretion system: improving capacity for secretion of recombinant protein.

Autor: Green CA; Integrated BioSciences, School of Clinical Dentistry, University of Sheffield, Sheffield, S10 2TA, UK.; Sustainable Process Technologies, Chemical and Environmental Engineering, University of Nottingham, Nottingham, NG7 2RD, UK., Kamble NS; Integrated BioSciences, School of Clinical Dentistry, University of Sheffield, Sheffield, S10 2TA, UK., Court EK; Integrated BioSciences, School of Clinical Dentistry, University of Sheffield, Sheffield, S10 2TA, UK., Bryant OJ; Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, UK., Hicks MG; Integrated BioSciences, School of Clinical Dentistry, University of Sheffield, Sheffield, S10 2TA, UK., Lennon C; FUJIFILM Diosynth Biotechnologies, Belasis Avenue, Stockton-on-Tees, Billingham, TS23 1LH, UK., Fraser GM; Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, UK., Wright PC; School of Engineering, The Faculty of Science, Agriculture and Engineering, Newcastle University, Newcastle, NE1 7RU, UK., Stafford GP; Integrated BioSciences, School of Clinical Dentistry, University of Sheffield, Sheffield, S10 2TA, UK. g.stafford@sheffield.ac.uk.
Jazyk: angličtina
Zdroj: Microbial cell factories [Microb Cell Fact] 2019 Jan 18; Vol. 18 (1), pp. 10. Date of Electronic Publication: 2019 Jan 18.
DOI: 10.1186/s12934-019-1058-4
Abstrakt: Background: Many valuable biopharmaceutical and biotechnological proteins have been produced in Escherichia coli, however these proteins are almost exclusively localised in the cytoplasm or periplasm. This presents challenges for purification, i.e. the removal of contaminating cellular constituents. One solution is secretion directly into the surrounding media, which we achieved via the 'hijack' of the flagellar type III secretion system (FT3SS). Ordinarily flagellar subunits are exported through the centre of the growing flagellum, before assembly at the tip. However, we exploit the fact that in the absence of certain flagellar components (e.g. cap proteins), monomeric flagellar proteins are secreted into the supernatant.
Results: We report the creation and iterative improvement of an E. coli strain, by means of a modified FT3SS and a modular plasmid system, for secretion of exemplar proteins. We show that removal of the flagellin and HAP proteins (FliC and FlgKL) resulted in an optimal prototype. We next developed a high-throughput enzymatic secretion assay based on cutinase. This indicated that removal of the flagellar motor proteins, motAB (to reduce metabolic burden) and protein degradation machinery, clpX (to boost FT3SS levels intracellularly), result in high capacity secretion. We also show that a secretion construct comprising the 5'UTR and first 47 amino acidsof FliC from E. coli (but no 3'UTR) achieved the highest levels of secretion. Upon combination, we show a 24-fold improvement in secretion of a heterologous (cutinase) enzyme over the original strain. This improved strain could export a range of pharmaceutically relevant heterologous proteins [hGH, TrxA, ScFv (CH 2 )], achieving secreted yields of up to 0.29 mg L -1 , in low cell density culture.
Conclusions: We have engineered an E. coli which secretes a range of recombinant proteins, through the FT3SS, to the extracellular media. With further developments, including cell culture process strategies, we envision further improvement to the secreted titre of recombinant protein, with the potential application for protein production for biotechnological purposes.
Databáze: MEDLINE
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