Vilnius University, Institute of Biotechnology, Department of Eukaryote Genetic Engineering, Graiciuno 8, LT-02241 Vilnius, Lithuania.
Microb Cell Fact. 2011 Dec 15;10:109. doi: 10.1186/1475-2859-10-109.
Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis.
The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody.
Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of pseudotype VLPs was successful and allowed generation of multivalent scFv-Fc proteins with high VLY-neutralizing potency. Our study demonstrated for the first time that large recombinant antibody molecule fused with hamster polyomavirus VP2 protein and co-expressed with VP1 protein in the form of pseudotype VLPs was properly folded and exhibited strong antigen-binding activity. The current study broadens the potential of recombinant VLPs as a highly efficient carrier for functionally active complex proteins.
重组抗体可以采用不同的形式和表达系统进行生产。单链可变片段（scFv）是全长抗体的一种有吸引力的替代物，它们可以很容易地在细菌或酵母中产生。然而，scFv 表现出单价抗原结合特性和较短的血清半衰期。scFv 的稳定性和亲和力可以通过其多聚化或与 IgG Fc 结构域融合来提高。本研究的目的是研究在酵母中生产高亲和力 scFv-Fc 蛋白以中和阴道加德纳菌主要毒力因子溶细胞素（VLY）的可能性。
从产生针对 VLY 的高亲和力中和抗体的杂交瘤细胞系衍生的 scFv 蛋白与人类 IgG1 Fc 结构域融合。构建并在酿酒酵母中生产了四种不同的抗 VLY scFv-Fc 融合蛋白变体。未标记的 scFv-Fc 和六组氨酸标记的 scFv-Fc 蛋白主要作为不溶性聚集体存在，因此不适合进一步纯化和活性测试。添加酵母α因子信号序列并没有支持抗 VLY scFv-Fc 的分泌，但增加了其细胞内可溶性形式的量。然而，纯化的蛋白显示出较弱的 VLY 中和能力。相比之下，抗 VLY scFv-Fc 分子与仓鼠多瘤病毒衍生的 VP2 蛋白融合及其与 VP1 蛋白的共表达导致了有效产生具有强 VLY 结合活性的假型病毒样颗粒（VLPs）。表达在 VLP 表面的重组 scFv-Fc 分子中和了 VLY 介导的人红细胞和 HeLa 细胞裂解，其效力与全长抗体相当。
重组 scFv-Fc 蛋白在酵母中的表达效率较低。将 scFv-Fc 分子展示在假型 VLPs 表面的新方法是成功的，并允许产生具有高 VLY 中和效力的多价 scFv-Fc 蛋白。我们的研究首次证明，与仓鼠多瘤病毒 VP1 蛋白共表达的融合有仓鼠多瘤病毒 VP2 蛋白的大型重组抗体分子以假型 VLPs 的形式正确折叠，并表现出强的抗原结合活性。本研究拓宽了重组 VLPs 作为功能活性复杂蛋白的高效载体的潜力。
