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Literature DB >> 28488120

Efficient Antibody Assembly in E. coli Periplasm by Disulfide Bond Folding Factor Co-expression and Culture Optimization.

Carlos Rodriguez^1,2, Dong Hyun Nam^1,3, Evan Kruchowy¹, Xin Ge⁴.

Abstract

Molecular chaperones and protein folding factors of bacterial periplasmic space play important roles in assisting disulfide bond formation and proper protein folding. In this study, effects of disulfide bond protein (Dsb) families were investigated on assembly of 3F3 Fab, an antibody inhibitor targeting matrix metalloproteinase-14 (MMP-14). By optimizing DsbA/C co-expression, promoter for 3F3 Fab, host strains, and culture media and conditions, a high yield of 30-mg purified 3F3 Fab per liter culture was achieved. Produced 3F3 Fab exhibited binding affinity of 34 nM and inhibition potency of 970 nM. This established method of DsbA/C co-expression can be applied to produce other important disulfide bond-dependent recombinant proteins in E. coli periplasm.

Entities: CellLine Chemical Disease Gene Species

Keywords: DsbA; DsbC; Fab; IgG; Over-expression; Periplasm; Protein folding factor

Mesh：

Substances：

Year: 2017 PMID： 28488120 PMCID： PMC5898612 DOI： 10.1007/s12010-017-2502-8

Source DB: PubMed Journal: Appl Biochem Biotechnol ISSN： 0273-2289 Impact factor: 2.926

24 in total

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Journal: J Mol Biol Date: 2011-11-27 Impact factor: 5.469

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Authors: Anton V Bryksin; Ichiro Matsumura
Journal: Biotechniques Date: 2010-06 Impact factor: 1.993

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Authors: Yariv Mazor; Thomas Van Blarcom; Robert Mabry; Brent L Iverson; George Georgiou
Journal: Nat Biotechnol Date: 2007-04-15 Impact factor: 54.908

6. Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones.

Authors: R Levy; R Weiss; G Chen; B L Iverson; G Georgiou
Journal: Protein Expr Purif Date: 2001-11 Impact factor: 1.650

Efficient Antibody Assembly in E. coli Periplasm by Disulfide Bond Folding Factor Co-expression and Culture Optimization.

1. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm.

2. Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies.

3. A reverse binding motif that contributes to specific protease inhibition by antibodies.

4. Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids.

5. Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli.

6. Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones.

7. Expression of active human tissue-type plasminogen activator in Escherichia coli.

8. A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases.

9. Quality control in the bacterial periplasm.

10. Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli.

1. Disulfide Chaperone Knockouts Enable In Vivo Double Spin Labeling of an Outer Membrane Transporter.

Review 2. Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments.