Huang Dagang, Shusta Eric V
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Wisconsin 53706, USA.
Biotechnol Prog. 2005 Mar-Apr;21(2):349-57. doi: 10.1021/bp0497482.
Green fluorescent protein (GFP) continues to be a very useful tool in biotechnology, but soluble production of GFP and GFP-protein fusions has been difficult. In this study, we have produced yeast-enhanced green fluorescent protein (yEGFP) in Saccharomyces cerevisiae as a soluble, secreted product with a purified level of 6 mg/L. Expression was directed by the inducible GAL1-10 promoter and synthetic prepro leader sequence. The secretion of yEGFP by yeast was strongly dependent on temperature, with 20 degrees C induction being optimal. Use of 2 micro multicopy expression constructs elevated yields over a low-copy CEN-based system by approximately 2-fold. Yeast-enhanced GFP was also expressed as a fusion to the Aga2p mating agglutinin in order to test the secretory processing fidelity of yEGFP-protein fusions. When the cell surface anchoring protein, Aga1p, was co-overexpressed with the Aga2p-yEGFP fusion, the Aga2p-yEGFP protein was tethered to the yeast cell surface. Flow cytometry and fluorescence microscopy analysis indicated that the fusion was displayed on the yeast cell surface at high levels. In the absence of high level Aga1p expression, the Aga2p-yEGFP fusion protein was instead secreted in its entirety with no detectable surface display. These findings reveal that yeast is a suitable host for secretion of GFP and GFP-protein fusions and thus could enable a wide range of biochemistry and biotechnology applications.
绿色荧光蛋白（GFP）仍然是生物技术中非常有用的工具，但GFP及其与蛋白质的融合体的可溶性表达一直很困难。在本研究中，我们在酿酒酵母中生产了酵母增强型绿色荧光蛋白（yEGFP），作为一种可溶性分泌产物，纯化水平为6 mg/L。表达由可诱导的GAL1-10启动子和合成的前原导序列指导。酵母对yEGFP的分泌强烈依赖于温度，20℃诱导最为适宜。使用2μm多拷贝表达构建体比基于低拷贝CEN的系统产量提高了约2倍。酵母增强型GFP还作为与Aga2p交配凝集素的融合蛋白表达，以测试yEGFP-蛋白质融合体的分泌加工保真度。当细胞表面锚定蛋白Aga1p与Aga2p-yEGFP融合体共同过表达时，Aga2p-yEGFP蛋白被拴系在酵母细胞表面。流式细胞术和荧光显微镜分析表明，该融合体在酵母细胞表面高水平展示。在没有高水平Aga1p表达的情况下，Aga2p-yEGFP融合蛋白则完全分泌，没有可检测到的表面展示。这些发现表明，酵母是分泌GFP及其与蛋白质融合体的合适宿主，因此可实现广泛的生物化学和生物技术应用。
