SORTING OF TRANSFECTED BACTERIA EXPRESSING GREEN FLUORESCENT PROTEIN

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.