CRYSTALLIZATION AND 3D STRUCTURE DETERMINATION OF B-1,4GALACTOSYLTRANSFERASE

B-1,4 半乳糖基转移酶的结晶和 3D 结构测定

• 批准号: 2463784
• 负责人: P K QASBA
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2463784
• 关键词: Escherichia coli; X ray crystallography; active sites; crystallization; enzyme activity; enzyme structure; galactosyltransferases; inclusion body; lactalbumin; method development; recombinant proteins; site directed mutagenesis; solubility

We have separately expressed the catalytic domain (R3) of beta-1,4 galactosyltransferase (beta-1,4 GT) and the full length enzyme, containing the transmembrane domain, in E.coli. The recombinant proteins are present in inclusion bodies as insoluble precipitates which do not exhibit any enzymatic activity. Previously a method was developed in this laboratory to generate soluble and active protein from the inclusion bodies. This method has been modified and improved to increase the solubility of the protein that is required for the crystallization purposes. Using this methods we are now able to produce enough quantity of R3 protein for crystallization studies. Preliminary crystallization studies indicate that the R3 protein crystals can be obtained in a period of 1 to 3 days. Attempts are being made to grow better quality and suitable size crystals for x-ray crystallographic investigations. To further improve the solubility of the recombinant protein, Phe residues, which might contribute to the low solubility were mutated. By site directed mutagenesis all Phe residues were mutated to Ala, one residue at a time and the mutated protein tested for the solubility. The results indicate that replacing the Phe residue at 149 or 226 or 236 with Ala, the solubility increases by three fold. However, when Phe at 149 is replaced by Lys there was no change in solubility. Further detailed investigations are being carried out. Similar to beta-1,4 GT, large amount of recombinant mouse alpha-lactalbumin, a modifier protein of beta-1,4 GT, is obtained as inclusion body in E.coli. We are able to produce large quantities of active and soluble protein from the inclusion bodies, using the method developed for beta-1,4 GT with a slight modification. Preliminary crystallization studies indicate that this protein crystallizes at room temperature using the conditions quite similar to the one used for human alpha-lactalbumin.

我们分别表达了β-1，4的催化结构域（R3） 半乳糖基转移酶（β-1，4 GT）和全长酶， 含有跨膜结构域。 重组 蛋白质作为不溶性沉淀物存在于包涵体中 其不显示任何酶活性。 以前有一种方法， 在这个实验室开发的， 从包涵体中分离出来。 该方法已被修改和改进 为了增加蛋白质的溶解度， 结晶的目的。 使用这些方法，我们现在可以 产生足够量的R3蛋白用于结晶研究。 初步的结晶研究表明，R3蛋白 可以在1至3天的时间内获得晶体。 尝试 使其生长更好的质量和适合X射线的尺寸的晶体 晶体学研究 为了进一步提高重组蛋白的溶解性， 突变可能导致低溶解度的残基。 通过定点诱变，将所有Phe残基突变为Ala， 一次检测一个残基，并测试突变蛋白的溶解度。 结果表明，取代149或226或228位的Phe残基， 236与Ala，溶解度增加三倍。 然而当 149位的Phe被Lys替代，溶解度没有变化。 正在进行进一步的详细调查。 类似于 β-1，4 GT，大量重组小鼠α-乳白蛋白，a β-1，4 GT的修饰蛋白，以包涵体形式获得， E.coli. 我们能够生产大量的活性和可溶性 蛋白质的包涵体，使用开发的方法， beta-1，4 GT略有改动 初步结晶 研究表明这种蛋白质在室温下结晶 使用与用于人类的条件非常相似的条件， α-乳白蛋白。