SORTING OF TRANSFECTED BACTERIA EXPRESSING GREEN FLUORESCENT PROTEIN

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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. 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.