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Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach
Published on: October 16, 2014
Secretion from bacterial versus mammalian cells yields a recombinant scFv with variable folding properties
Michelle C Vendel1, Michael Favis, William B Snyder
1Biogen Idec, 14 Cambridge Center, Cambridge, MA 02142, USA.
Archives of Biochemistry and Biophysics
|January 11, 2012
Summary
Bacterially expressed single-chain variable fragments (scFvs) can misfold, leading to challenging antigen binding. Careful characterization of bacterial scFv folding is crucial for antibody engineering, especially when non-specific binding occurs.
Area of Science:
- Biotechnology
- Protein Engineering
- Immunology
Background:
- Escherichia coli (E. coli) is widely used for expressing antibody fragments like single-chain variable fragments (scFvs).
- Previous work focused on expressing well-folded scFvs in E. coli for antibody engineering, aiming for bispecific antibodies (BsAbs).
Purpose of the Study:
- To investigate the structural differences of scFvs expressed in E. coli versus Chinese Hamster Ovary (CHO) cells.
- To compare the properties of E. coli-derived scFvs with those derived from CHO cells as part of a BsAb.
Main Methods:
- Expressed and secreted an scFv as part of a BsAb in CHO cells.
- Developed a proteolytic method to isolate the CHO-derived scFv.
- Compared the structural and binding properties of E. coli-derived and CHO-derived scFvs.
Main Results:
- E. coli-expressed scFvs were found in a stable, misfolded monomeric state, unlike CHO-derived scFvs.
- The misfolded E. coli scFv exhibited heterogeneous and non-specific antigen binding, complicating characterization.
- A robust proteolytic method was established for obtaining intact scFvs from CHO-expressed IgG-like antibodies.
Conclusions:
- Bacterial expression of scFvs can lead to misfolding, necessitating thorough characterization of folded properties.
- The developed proteolytic method provides a reliable way to produce scFvs from CHO cell lines without specific cleavage sites.
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