Gąciarz, A., Khatri, N., Velez-Suberbie, M., Saaranen, M., Uchida, Y., Keshavarz-Moore, E., Ruddock, L. (2017) Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media. Microbial Cell Factories, 16, 108. doi:10.1186/s12934-017-0721-x
Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media
|Author:||Gąciarz, Anna1; Khatri, Narendar Kumar2; Velez-Suberbie, M. Lourdes3;|
1Faculty of Biochemistry and Molecular Medicine, University of Oulu
2The Department of Process and Environment Engineering, University of Oulu
3The Advanced Center for Biochemical Engineering, Department of Biochemical Engineering, University College London
|Online Access:||PDF Full Text (PDF, 1.5 MB)|
|Persistent link:|| http://urn.fi/urn:nbn:fi-fe201902205798
|Publish Date:|| 2019-02-20
Background: The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm.
Results: Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source.
Conclusions: Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.
Microbial cell factories
|Type of Publication:||
A1 Journal article – refereed
|Field of Science:||
318 Medical biotechnology
This work was supported by funding from the Academy of Finland, Sigrid Juselius Foundation and Biocenter Oulu.
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