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
Department of Aquaculture, National Kaohsiung Marine University, Kaohsiung, Taiwan.
Department of Biological Science and Technology, National Chiao Tung University, Hsin-Chu, Taiwan.
PLoS One. 2014 May 13;9(5):e97367. doi: 10.1371/journal.pone.0097367. eCollection 2014.
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.
单链可变片段（scFv）可替代全长单克隆抗体，用于研究、治疗及诊断应用。然而，当重组scFv在细菌中过表达时，它们常常形成包涵体并丧失功能。为克服这一问题，我们开发了一种scFv分泌系统，其中scFv与渗透诱导蛋白Y（osmY，一种细菌分泌载体蛋白）融合，以实现高效的蛋白质分泌。抗表皮生长因子受体scFv（αEGFR）分别与osmY（N端和C端）及周质前导序列（pelB）融合，以产生αEGFR-osmY、osmY-αEGFR和pelB-αEGFR（对照）。与对照相比，两种与osmY融合的αEGFR scFv均为可溶性，并分泌到LB培养基中。此外，可溶性αEGFR-osmY的产量高20倍，分泌蛋白的量比osmY-αEGFR高250倍。此外，两种与osmY融合的αEGFR的抗原结合活性比从包涵体中复性的pelB-αEGFR高2倍。在αTAG72-osmY和αHer2-osmY中也观察到了类似的结果。这些结果表明，与scFv融合时，osmY的N端可产生高产率的可溶性、功能性及分泌性scFv，基于osmY的细菌分泌系统可用于低成本αEGFR蛋白的大规模工业化生产。
