標題: Functional Production of a Soluble and Secreted Single-Chain Antibody by a Bacterial Secretion System
作者: Cheng, Chiu-Min
Tzou, Shey-Cherng
Zhuang, Ya-Han
Huang, Chien-Chiao
Kao, Chien-Han
Liao, Kuang-Wen
Cheng, Ta-Chun
Chuang, Chih-Hung
Hsieh, Yuan-Chin
Tai, Ming-Hong
Cheng, Tian-Lu
生物科技學系
Department of Biological Science and Technology
公開日期: 13-五月-2014
摘要: 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 (alpha EGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate alpha EGFR-osmY, osmY-alpha EGFR, and pelB-alpha EGFR (control), respectively. In comparison with the control, both the osmY-fused alpha EGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble alpha EGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-alpha EGFR. In addition, the antigenbinding activity of both the osmY-fused alpha EGFRs was 2-fold higher than that of the refolded pelB-alpha EGFR from inclusion bodies. Similar results were observed with alpha TAG72-osmY and alpha Her2-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 alpha EGFRprotein.
URI: http://dx.doi.org/10.1371/journal.pone.0097367
http://hdl.handle.net/11536/24424
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0097367
期刊: PLOS ONE
Volume: 9
Issue: 5
結束頁: 
顯示於類別:期刊論文


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