Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system

Chiu-Min Cheng; Shey-Cherng Tzou; Ya-Han Zhuang; Chien-Chiao Huang; Chien-Han Kao; Kuang-Wen Liao; Ta-Chun Cheng; Chih-Hung Chuang; Yuan-Chin Hsieh; Ming-Hong Tai; Tian-Lu Cheng

doi:10.1371/journal.pone.0097367

Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system

PLoS One. 2014 May 13;9(5):e97367. doi: 10.1371/journal.pone.0097367. eCollection 2014.

Authors

Affiliations

¹ Department of Aquaculture, National Kaohsiung Marine University, Kaohsiung, Taiwan.
² Department of Biological Science and Technology, National Chiao Tung University, Hsin-Chu, Taiwan.
³ Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁴ Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁵ Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
⁶ Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan; Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan; Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.

Abstract

Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibodies used in research and therapeutic and diagnostic applications. However, when recombinant scFvs are overexpressed in bacteria, they often form inclusion bodies and exhibit loss of function. To overcome this problem, we developed an scFv secretion system in which scFv was fused with osmotically inducible protein Y (osmY), a bacterial secretory carrier protein, for efficient protein secretion. Anti-EGFR scFv (αEGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR (control), respectively. In comparison with the control, both the osmY-fused αEGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble αEGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-αEGFR. In addition, the antigen-binding activity of both the osmY-fused αEGFRs was 2-fold higher than that of the refolded pelB-αEGFR from inclusion bodies. Similar results were observed with αTAG72-osmY and αHer2-osmY. These results suggest that the N-terminus of osmY fused with scFv produces a high yield of soluble, functional, and secreted scFv, and the osmY-based bacterial secretion system may be used for the large-scale industrial production of low-cost αEGFR protein.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacterial Secretion Systems / immunology*
Bioreactors*
Blotting, Western
DNA Primers / genetics
Enzyme-Linked Immunosorbent Assay
ErbB Receptors / metabolism
Escherichia coli / genetics
Escherichia coli / metabolism*
Escherichia coli Proteins / metabolism*
Industrial Microbiology / methods*
Periplasmic Binding Proteins / metabolism*
Protein Sorting Signals / genetics
Single-Chain Antibodies / biosynthesis*
Single-Chain Antibodies / immunology
Single-Chain Antibodies / metabolism

Substances

Bacterial Secretion Systems
DNA Primers
Escherichia coli Proteins
Periplasmic Binding Proteins
Protein Sorting Signals
Single-Chain Antibodies
osmY protein, E coli
ErbB Receptors

Grants and funding

This work was supported by grants from the National Research Program for Biopharmaceuticals, National Science Council, Taipei, Taiwan (NSC101-2325-B-037- 001, NSC101-2321-B-037-001, NSC101-2313-B-022-001), the Department of Health, Executive Yuan, Taiwan (DOH100-TD-C-111-002) and the Grant of Biosignature in Colorectal Cancers, Academia Sinica, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.