Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae
Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae
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DOI：
10.1128/aem.01466-14
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发表时间：
2014-09-01
影响因子：
4.4
通讯作者：
Shusta, Eric V.
中科院分区：
文献类型：
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作者：
Burns, Michael L.;Malott, Thomas M.;Shusta, Eric V.
脑源性神经营养因子（BDNF）在神经系统功能中起重要作用，具有治疗潜力。BDNF的微生物生产已经导致低保真度蛋白质产物，通常以不能结合同源TrkB或p75受体的大的不溶性聚集体的形式。在这项研究中，采用酿酒酵母展示和分泌系统，它被发现，BDNF是穷人的表达和部分无活性的酵母表面和BDNF分泌在低水平的形式二硫键结合的聚集体。因此，为了增加酵母作为BDNF表达宿主的相容性，采用定向进化方法来改善BDNF折叠和表达水平。将酵母表面展示与两轮采用随机诱变和改组的定向进化相结合，以鉴定表达提高5倍、特异性TrkB结合活性增加4倍并恢复p75结合活性的BDNF突变体，所述突变体既作为展示蛋白又作为分泌蛋白。在转染的PC12细胞中，发现分泌的BDNF突变体主要以可溶性同源二聚体的形式存在，其可以刺激TrkB磷酸化。定点诱变研究表明，一个特别重要的突变类涉及引入半胱氨酸接近天然半胱氨酸，参与BDNF半胱氨酸结结构。总之，这些发现表明酵母现在是BDNF生产和工程的可行替代品。
Brain-derived neurotrophic factor (BDNF) plays an important role in nervous system function and has therapeutic potential. Microbial production of BDNF has resulted in a low-fidelity protein product, often in the form of large, insoluble aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, employing Saccharomyces cerevisiae display and secretion systems, it was found that BDNF was poorly expressed and partially inactive on the yeast surface and that BDNF was secreted at low levels in the form of disulfide-bonded aggregates. Thus, for the purpose of increasing the compatibility of yeast as an expression host for BDNF, directed-evolution approaches were employed to improve BDNF folding and expression levels. Yeast surface display was combined with two rounds of directed evolution employing random mutagenesis and shuffling to identify BDNF mutants that had 5-fold improvements in expression, 4-fold increases in specific TrkB binding activity, and restored p75 binding activity, both as displayed proteins and as secreted proteins. Secreted BDNF mutants were found largely in the form of soluble homodimers that could stimulate TrkB phosphorylation in transfected PC12 cells. Site-directed mutagenesis studies indicated that a particularly important mutational class involved the introduction of cysteines proximal to the native cysteines that participate in the BDNF cysteine knot architecture. Taken together, these findings show that yeast is now a viable alternative for both the production and the engineering of BDNF.