An Improved Single-Chain Fab Platform for Efficient Display and Recombinant Expression. Issue 2 (30th January 2015)

• Record Type: Journal Article
• Authors: Koerber, James T.; Hornsby, Michael J.; Wells, James A.
• Is Part Of: Journal of molecular biology. Volume 427:Issue 2(2015:Jan. 15)
• Page Start: 576
• Page End: 586
• Publication Date: 2015-01-30
• Subjects: Fab fragment antigen binding -- scFv single-chain fragment variable -- scFab single-chain Fab -- scIgG single-chain IgG -- CDR complementarity-determining region -- SRP signal recognition particle -- HPep human anti-peptide -- DSF differential scanning fluorimetry -- HRP horseradish peroxidase -- PBS phosphate-buffered saline; antibody -- signal peptide -- high-throughput screening -- phage display -- bacterial and mammalian expression; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2014.11.017
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however, many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain fragment antigen binding (Fab) (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain fragment variable with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1–3 mg/L. Furthermore, rerouting of the scFab to the co-translational signal recognition particle pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable ( T m of 81 °C), predominantly monomeric (> 90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies. Graphical abstract: Highlights: High-throughput antibody generation requires improved scaffolds. Improved phage display for a scFab with long linkers and optimized signal peptide. Rapid reformatting for high yield, stable scFab from bacterial and mammalian hosts. New, optimized scFab to advance antibody discovery.