Design and analysis of biomolecules based on library screening

（1）文库筛选对于筛选的第二步，建立实验系统以在大肠杆菌细胞中表达具有以下重复序列的蛋白质文库：（Gly-Gly-X1-X2-X3-Gly-X4-Pro-X5-X6-Asn-Ala）n，其中X1、IIe、Leu、瓦尔或Phe ; X2、Ser或Thr ; X3、IIe、Leu、瓦尔或Phe ; X4、Ala或Gly ; X5、Arg或His ; X6、Glu或瓦尔;筛选文库后，得到9个可溶性蛋白表达克隆，并测定了它们的核苷酸序列。这些克隆中的一些产生可溶性蛋白，重复次数>50。（2）可溶性蛋白的纯化在蛋白质的C端加入His标签序列，通过His结合层析和羟基磷灰石层析进行纯化。（3）蛋白质的结构分析及功能研究通过圆二色谱分析纯化蛋白质的结构，发现这些蛋白质具有无规卷曲结构。这些具有重复序列的蛋白质被设计成具有规则间隔的疏水残基。因此，蛋白质可以结合具有重复结构的小疏水分子。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起结构变化。

(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.