SORTING OF TRANSFECTED BACTERIA EXPRESSING GREEN FLUORESCENT PROTEIN

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 本项目的目标是鉴定分子进化后表达可溶性荧光蛋白的细菌，以改善蛋白质的折叠和溶解性，用于蛋白质结构测定。 结构感兴趣的蛋白质将与修饰的EGFP报告基因融合，并通过DNA改组和易错PCR进行分子进化。 将所得文库转染到细菌中并表达。 由于融合构建体的适当折叠是EGFP荧光所需的，因此表达可溶性蛋白的细菌将是明亮的。 然后克隆分选的细菌以分离正确折叠的蛋白质的单个变体用于晶体学研究。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this project is to identify bacteria expressing soluble fluorescent protein after moleulcar evolution to improve protein folding and solubility for protein structure determination. Proteins whose structure is of interest will be genetically fused to a modified EGFP reporter and subjected to molecular evolution by DNA shuffling and error prone PCR. The resulting library will be transfeected into bacteria and expressed. Because the proper folding of the fusion construct is required for EGFP fluorescence, bacteria expressing soluble protein will be bright. Sorted bacteria are then cloned to isolate single variants of properly folded protein for crystallographic studies.