Development of selenomethionine resistant yeast

描述(申请人提供)：越来越多的基因组的完成推动了结构基因组学领域的发展，目标是在未来几年内确定数千个结构。这需要对晶体结构测定中的每一步进行实质性的优化。在这方面，目前常规使用的最重要的技术之一是生产硒蛋氨酸(SeMet)取代的重组蛋白。在某些方面，SEMET推动了结晶学领域的发展，因为聚合酶链式反应(PCR)促进了分子生物学的发展。SEMET有助于在多肽链中定位蛋氨酸，以及通过多波长异常色散(MAD)进行相确定，从而实现更有效的结构确定。SEMET通常用于表达来自大肠杆菌的重组蛋白，但随着完成的真核基因组数量的增加，在真核细胞中表达的需求越来越大。酿酒酵母提供了强大的遗传系统和高水平的内源和外源真核蛋白的生产，而且成本低，易于生长。在酵母中常规生产用于高分辨率结构分析的完全取代的SeMet蛋白是远远不能实现的。这项高风险/高影响建议的目标是将酵母遗传学和硒生物学方面的专业知识结合起来，开发用于生产用于结构分析的SEMET蛋白的酵母菌株。已确定的赋予SeMet耐药性的因素将进一步提供对硒毒性生物学的洞察。此外，用于产生SEMET抗性的筛选将导致鉴定基因产物，这些基因产物也可以被毕赤酵母或杆状病毒感染的细胞靶向，以增强对SEMET的耐受性，从而在这些系统产生的蛋白质中占据位置。通过增加本建议中描述的可用选择库，我们可以最大限度地筛选高通量结构方法的蛋白质生产，利用许多可用的克隆和标记的酵母蛋白质来增强我们对蛋白质功能的分子理解，进一步优化其他真核表达系统以用于SEMET的掺入，并更好地了解硒的毒性。

DESCRIPTION (provided by applicant): The completion of a growing number of genomes has driven the structural genomics field with the aim of determining many thousand structures within the next few years. This requires substantial optimization of every step in a crystallographic structure determination. In this aspect, one of the most important techniques now routinely used is the production of selenomethionine (SeMet) substituted recombinant proteins. In some ways SeMet has advanced the crystallography field as polymerase chain reaction (PCR) has advanced molecular biology. SeMet aids in positioning methionines in the polypeptide chain as well as for phase determination by multiwavelength anomalous dispersion (MAD) allowing more efficient structure determinations. SeMet is typically utilized for expression of recombinant proteins from Escherichia coli, but there is a growing need for expression in eukaroyotic cells as the number of eukaryotic genomes completed increases. The yeast Saccharomyces cerevisiae offers powerful genetic systems and high level production of endogenous and exogenous eukaryotic proteins with low cost and ease of growth. The routine production of completely substituted SeMet proteins for high resolution structural analysis is far from achieved in yeast. The goals of this high risk/high impact proposal are to bring together expertise in yeast genetics and selenium biology to develop yeast strains for the production of SeMet proteins for structural analysis. The factors identified to confer SeMet resistance will further provide insight into the biology of selenium toxicity. Additionally, the screens utilized to develop SeMet resistance will result in the identification of gene products that can also be targeted in Pichia pastoris or baculovirus infected cells to enhance SeMet tolerance and thus occupancy in the proteins produced from these systems. By increasing our repertoire of available choices as described in this proposal we can maximize the ability to screen protein production for high throughput structural approaches, utilize the many available cloned and tagged yeast proteins for enhancing our molecular understanding of protein function, further optimize other eukaryotic expression systems for SeMet incorporation and develop a better understanding of Se toxicity.