Ye Tingmei, Lin Zhihua, Lei Huanzong
Department of Biology, School of Chemistry and Life Sciences, Lishui University, Lishui, 323000, Zhejiang, People's Republic of China.
Appl Microbiol Biotechnol. 2008 Nov;81(2):311-7. doi: 10.1007/s00253-008-1655-3. Epub 2008 Sep 16.
Antibodies currently constitute the most rapidly growing class of human therapeutics; however, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. High expression of active single-chain antibody fragment (scFv) in Escherichia coli has not been successful, as the protein contains three intramolecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the scFv gene against VEGF165 with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-scFv fusion gene that was highly expressed in the BL21(DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-scFv, was up to 28.5% of the total cellular protein. The fusion protein was purified by Ni nitrilotriacetic acid (NTA) affinity chromatography and cleaved by a SUMO-specific protease to obtain the native scFv, which was further purified by Ni-NTA affinity chromatography. The result of the high-performance liquid chromatography showed that the purity of the recombinant cleaved scFv was greater than 98%. The primary structure of the purified scFv was confirmed by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analysis. In vitro activity assay demonstrated that the recombinant scFv could dose-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cell proliferation. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant scFv with native sequences.
抗体目前是人类治疗药物中增长最为迅速的一类；然而，重组抗体和抗体片段的高产制备是一项切实的挑战。活性单链抗体片段（scFv）在大肠杆菌中的高表达尚未成功，因为该蛋白含有三个分子内二硫键，难以在细菌细胞内环境中正确形成。为解决这一问题，我们通过合成在BL21(DE3)菌株中高表达的人工SUMO-scFv融合基因，将抗VEGF165的scFv基因与一个小泛素相关修饰基因（SUMO）融合。可溶性融合蛋白SUMO-scFv的最佳表达水平高达总细胞蛋白的28.5%。融合蛋白通过镍次氮基三乙酸（NTA）亲和层析进行纯化，并用SUMO特异性蛋白酶进行切割以获得天然scFv，再通过镍- NTA亲和层析进一步纯化。高效液相色谱结果表明，重组切割后的scFv纯度大于98%。通过N端氨基酸测序和基质辅助激光解吸/电离飞行时间质谱分析确认了纯化scFv的一级结构。体外活性测定表明，重组scFv能够剂量依赖性地抑制VEGF165诱导的人脐静脉来源内皮细胞增殖。本研究提出的表达策略能够方便地实现重组scFv的高产和易于纯化，且具有天然序列。
