Huang Dagang, Shusta Eric V
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
Appl Environ Microbiol. 2006 Dec;72(12):7748-59. doi: 10.1128/AEM.01403-06. Epub 2006 Oct 6.
Fusion proteins comprised of a binding domain and green fluorescent protein (GFP) have the potential to act as one-step binding reagents. In this study, eight single-chain antibodies (scFv) and one single-chain T-cell receptor (scTCR) were secreted as fusions to GFP using a Saccharomyces cerevisiae expression system. Fusion protein secretion levels ranged over 3 orders of magnitude, from 4 microg/liter to 4 mg/liter, and correlated well with the secretion levels of the unfused scFv/scTCR. Three fusion types with various linker lengths and fusion orientations were tested for each scFv/scTCR. Although the fusion protein secretion levels were not significantly affected by the nature of the fusion construct, the properties of the fusion protein were clearly influenced. The fluorescence yield per fusion molecule was increased by separating the scFv/scTCR and GFP with an extended (GGGGS)3 linker, and fusions with scFv/scTCR at the carboxy-terminus were more resistant to degradation. By evaluating leader sequence processing and using GFP fluorescence to track intracellular processing, it was determined that the majority of fusion protein synthesized by the yeast was not secreted and in most cases was accumulating in an immature, although active, endoplasmic-reticulum (ER)-processed form. This contrasted with unfused scFv, which accumulated in both immature ER-processed and mature post-Golgi forms. The results indicated that yeast can be used as an effective host for the secretion of scFv/scTCR-GFP fusion proteins and that as a result of intracellular secretory bottlenecks, there is considerable yeast secretory capacity remaining to be exploited.
由结合结构域和绿色荧光蛋白（GFP）组成的融合蛋白有潜力作为一步结合试剂。在本研究中，使用酿酒酵母表达系统分泌了8种单链抗体（scFv）和1种单链T细胞受体（scTCR）与GFP的融合蛋白。融合蛋白的分泌水平跨越3个数量级，从4微克/升至4毫克/升，并且与未融合的scFv/scTCR的分泌水平高度相关。针对每种scFv/scTCR测试了三种具有不同接头长度和融合方向的融合类型。尽管融合蛋白的分泌水平不受融合构建体性质的显著影响，但融合蛋白的性质明显受到影响。通过用延长的（GGGGS）3接头分离scFv/scTCR和GFP，每个融合分子的荧光产量增加，并且在羧基末端与scFv/scTCR融合的蛋白对降解更具抗性。通过评估前导序列加工并使用GFP荧光追踪细胞内加工过程，确定酵母合成的大多数融合蛋白未被分泌，并且在大多数情况下以未成熟但有活性的内质网（ER）加工形式积累。这与未融合的scFv形成对比，后者以未成熟的ER加工形式和成熟的高尔基体后形式积累。结果表明，酵母可作为分泌scFv/scTCR-GFP融合蛋白的有效宿主，并且由于细胞内分泌瓶颈，仍有相当大的酵母分泌能力有待开发。
