Design and analysis of biomolecules based on library screening

(1)文库筛选对于第二步的筛选，建立了一个在大肠杆菌细胞中表达蛋白质的实验系统，该文库具有以下重复序列：(Gly-Gly-X1-X2-X3-Gly-X4-Pro-X5-X6-Asn-Ala)n，：X1、IIe、Leu、Val或Phe；X2、Ser或Thr；X3、IIe、Leu、Val或Phe；X4、Ala或Gly；X5、Arg或His；X6、Glu或Val；n&gt；10.通过对文库的筛选，获得了9个表达可溶性蛋白的克隆，并测定了它们的核苷酸序列。(2)可溶性蛋白的纯化将His-tag序列添加到蛋白质的C端，然后用His-Bind层析和羟基磷灰石层析进行纯化。(3)蛋白质的结构分析和可能的功能探索用圆二色谱对纯化的蛋白质进行结构分析，发现这些蛋白质具有随机卷曲结构。这些具有重复序列的蛋白质被设计成以规则的间隔具有疏水残基。因此，蛋白质有可能与具有重复结构的疏水小分子结合。我现在正在研究蛋白质是否与它们结合，以及结合这些小分子是否会引发结构变化。

(1)Library screeningFor the second step of the screening, an experimental system was established to express proteins in E coli cells a library of proteins with the following repetitive sequences :(Gly-Gly-X1-X2-X3-Gly-X4-Pro-X5-X6-Asn-Ala)n,where X1,IIe,Leu,Val, or Phe ; X2,Ser or Thr ; X3,IIe,Leu,Val, or Phe ; X4,Ala or Gly ; X5, Arg or His ; X6,Glu or Val ; n>10.After screening the library, nine clones were found to express soluble proteins, and their nucleotide sequences were determined. Some of these clones produced soluble proteins with the number of repetition of>50.(2)Purification of the soluble proteinsThe His-tag sequence was added at the C termini of the proteins, and they were purified by His-bind chromatography followed by hydroxyapatite chromatography.(3)Structural analysis of the proteins and search for possible functionsThe structures of the purified proteins were analyzed by circular dichrosim spectroscopy measurements to find these proteins have random coil structures. These proteins with repetitive sequences were designed to have hydrophobic residues at regular intervals. Thus, it is possible the proteins can bind small hydrophobic molecules with repetitive structures. I am now studying if the proteins bind them and if structural changes are elicited upon binding these small molecules.