SORTING OF TRANSFECTED BACTERIA EXPRESSING GREEN FLUORESCENT PROTEIN

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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，用于蛋白质结构的测定。其结构感兴趣的蛋白质将在基因上与修改后的EGFP报告基因融合，并通过DNA改组和容易出错的PCR进行分子进化。得到的文库将被转入细菌中并进行表达。因为融合结构的正确折叠是EGFP荧光所必需的，所以表达可溶性蛋白的细菌将是光明的。分选的细菌随后被克隆，以分离正确折叠的蛋白质的单一变体，用于结晶学研究。

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.