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.

我们已经分别表达了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， 在大肠杆菌中，含有跨膜结构域。重组人 蛋白质以不溶沉淀物的形式存在于包涵体中。 它们不表现出任何酶活性。以前有一种方法是 在这个实验室中开发，以产生可溶性和活性蛋白质 从包涵体中提取。对该方法进行了改进和改进 以增加蛋白质的溶解度，这是 结晶目的。使用这种方法，我们现在能够 产生足够数量的R3蛋白用于结晶研究。 初步结晶研究表明，R3蛋白 晶体可以在1-3天的时间内获得。尝试次数为 用于生长质量更好、尺寸适合x射线的晶体 结晶学研究 为了进一步提高重组蛋白Phe的溶解性 可能导致低溶解度的残基发生了突变。 通过定点突变，所有的Phe残基都突变为Ala，1 一次检测残留物，并检测突变蛋白质的溶解性。 结果表明，将Phe残留量替换为149或226或 236加入丙氨酸后，其溶解度增加了3倍。但是，当 Phe在149位被Lys取代，溶解度没有变化。 进一步的详细调查正在进行中。类似于 β-1，4-GT，大量重组小鼠α-乳清蛋白，a β-1，4-GT修饰蛋白以包涵体形式在 大肠埃希菌。我们能够大量生产活性物质和可溶性物质 从包涵体中提取蛋白质，使用为 β-1，4 GT，稍加修改。初步结晶 研究表明，这种蛋白质在室温下结晶。 使用与人类使用的条件非常相似的条件 α-乳清蛋白。