Evaluation of in vitro refolding vs cold shock expression: Production of a low yielding single chain variable fragment

Vermeulen, Jan‐G; Burt, Felicity J.; Heerden, Esta van; Cason, Errol D.; Meiring, Muriel

doi:10.1016/j.pep.2018.06.005

Cited by 8 publications

(2 citation statements)

References 48 publications

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“…Although several methods for successful expression of human kinases in E . coli were reported in literature, there are few comparative studies between these methods for the same kinase [ 8 , 14 , 15 ]. In this work, we compare the expression of three diverse enzymes: the tyrosine kinase domain of Epidermal Growth Factor Receptor (EGFR-KD), the serine-threonine kinase domain of Aurora A (AurKA-KD), and the full-length mixed kinase Mitogen-activated protein Kinase Kinase (MKK3).…”

Section: Introductionmentioning

confidence: 99%

Comparative expression of soluble, active human kinases in specialized bacterial strains

et al. 2022

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Kinases act as molecular switches for cellular functions and are involved in multiple human pathogeneses, most notably cancer. There is a continuous need for soluble and active kinases for in-vitro drug discovery and structural biology purposes. Kinases remain challenging to express using Escherichia coli, the most widely utilized host for heterologous expression. In this work, four bacterial strains, BL21 (DE3), BL21 (DE3) pLysS, Rosetta, and Arctic Express, were chosen for parallel expression trials along with BL21 (DE3) complemented with folding chaperones DnaJ/K and GroEL/ES to compare their performance in producing soluble and active human kinases. Three representative diverse kinases were studied, Epidermal Growth Factor Receptor kinase domain, Aurora Kinase A kinase domain, and Mitogen-activated protein Kinase Kinase. The genes encoding the kinases were subcloned into pET15b bacterial plasmid and transformed into the bacterial strains. Soluble kinase expression was tested using different IPTG concentrations (1–0.05 mM) at varying temperatures (37°C– 10°C) and induction times (3–24 hours). The optimum conditions for each kinase in all strains were then used for 1L large scale cultures from which each kinase was purified to compare yield, purity, oligomerization status, and activity. Although using specialized strains achieved improvements in yield and/or activity for the three kinases, none of the tested strains was universally superior, highlighting the individuality in kinase expression.

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Section: Introductionmentioning

confidence: 99%

Comparative expression of soluble, active human kinases in specialized bacterial strains

et al. 2022

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“…Redox engineered E. coli SHuffle cells have been previously demonstrated to be an attractive platform for the expression of various antibody formats, such as full-length IgG (Reddy et al 2018 ; Robinson et al 2015 ), Fab’ fragments (Abe et al 2014 ; Mori et al 2018 ; Yusakul et al 2018 ), scFv chains (Ahmadzadeh et al 2020 ; Liu et al 2019 ; Vermeulen et al 2018 ) and VHH domains (Eliseev et al 2018 ; Ta et al 2015 ; Zarschler et al 2013 ). Although SHuffle cells lack the eukaryotic glycosylation machinery, by engineering mutations in the Fc region of antibodies, the requirement for glycosylation for efficient binding of the IgG to its cognate receptor was bypassed (Robinson et al 2015 ).…”

Section: Introductionmentioning

confidence: 99%

Improved production of Humira antibody in the genetically engineered Escherichia coli SHuffle, by co-expression of human PDI-GPx7 fusions

Lénon

Szady

et al. 2020

Appl Microbiol Biotechnol

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Microbial production of antibodies offers the promise of cheap, fast, and efficient production of antibodies at an industrial scale. Limiting this capacity in prokaryotes is the absence of the post-translational machinery, present in dedicated antibody producing eukaryotic cell lines, such as B cells. There has been few and limited success in producing full-length, correctly folded, and assembled IgG in the cytoplasm of prokaryotic cell lines. One such success was achieved by utilizing the genetically engineered Escherichia coli strain SHuffle with an oxidative cytoplasm. Due to the genetic disruption of reductive pathways, SHuffle cells are under constant oxidative stress, including increased levels of hydrogen peroxide (H2O2). The oxidizing capacity of H2O2 was linked to improved disulfide bond formation, by expressing a fusion of two endoplasmic reticulum-resident proteins, the thiol peroxidase GPx7 and the protein disulfide isomerase, PDI. In concert, these proteins mediate disulfide transfer from H2O2 to target proteins via PDI-Gpx7 fusions. The potential of this new strain was tested with Humira, a blockbuster antibody usually produced in eukaryotic cells. Expression results demonstrate that the new engineered SHuffle strain (SHuffle2) could produce Humira IgG four-fold better than the parental strain, both in shake-flask and in high-density fermentation. These preliminary studies guide the field in genetically engineering eukaryotic redox pathways in prokaryotes for the production of complex macromolecules. Key points • A eukaryotic redox pathway was engineered into the E. coli strain SHuffle in order to improve the yield of the blockbuster antibody Humira. • The best peroxidase-PDI fusion was selected using bioinformatics and in vivo studies. • Improved yields of Humira were demonstrated at shake-flask and high-density fermenters.

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Characterization of the inhibition mechanism of a tissuefactor inhibiting single-chain variable fragment: a combined computational approach

et al. 2020

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Evaluation of in vitro refolding vs cold shock expression: Production of a low yielding single chain variable fragment

Cited by 8 publications

References 48 publications

Comparative expression of soluble, active human kinases in specialized bacterial strains

Comparative expression of soluble, active human kinases in specialized bacterial strains

Improved production of Humira antibody in the genetically engineered Escherichia coli SHuffle, by co-expression of human PDI-GPx7 fusions

Characterization of the inhibition mechanism of a tissuefactor inhibiting single-chain variable fragment: a combined computational approach

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