短短芽孢杆菌GZDF3中PilZ结构域基因的挖掘和糖基转移酶的原核表达

Mining gene with PilZ domain and prokaryotic expression of glycosyltransferase of Brevibacillus brevis GZDF3

摘要:

【背景】PilZ结构域是最早发现的环二鸟苷酸(Cyclic diguanylate，c-di-GMP)受体信号分子，与c-di-GMP结合后可以调控目标基因或者蛋白的活性，在细菌的生长过程中发挥着至关重要的作用，而短短芽孢杆菌中PilZ结构域的研究相对缺乏。【目的】挖掘短短芽孢杆菌GZDF3菌株中的PilZ结构域蛋白基因，并进行重组表达，为研究其功能奠定基础。【方法】从Pfam数据库中下载PilZ结构域模型，HMMScan软件扫描GZDF3全基因组序列，在保守结构域数据库(Conserved domain database，CDD)中分析蛋白保守结构域，Protein BLAST比对分析；采用ExPASy在线软件预测蛋白的基本理化性质；构建重组表达载体进行蛋白重组表达。【结果】GZDF3基因中存在5个含有PilZ结构域的蛋白编码基因，其中命名为Gene4836的基因经Protein BLAST比对分析显示其编码糖基转移酶，Gene1423为YcgR超家族蛋白编码基因，Gene1723编码透明质酸合成酶，属于糖基转移酶超家族2，其余Gene2571、Gene2956编码假定蛋白；Gene4836的编码产物分子量为24.08 kD，等电点为6.39，为酸性亲水性蛋白；C端有一个PilZ结构域；0.5 mmol/L乳糖诱导、30 °C培养20 h，表达出一大小约为25 kD的重组蛋白，与生物信息学预测结果相符。【结论】首次对短短芽孢杆菌含有PilZ结构域蛋白编码基因进行原核表达，并成功纯化出重组蛋白，为后续研究其功能奠定了基础。

关键词:短短芽孢杆菌，PilZ结构域，糖基转移酶，原核表达，重组蛋白

Abstract:

[Background] PilZ domain is the earliest discovered cyclic diguanylate (c-di-GMP) receptor signaling molecule, regulates the activity of the target gene or protein after binding to c-di-GMP, and plays a vital role in the growth of bacteria. However, the study of PilZ domain in Brevibacillus breves is a relative deficiency. [Objective] To mine the gene of PilZ domain in Brevibacillus breves GZDF3 strain and construct the recombinant expression system for further research. [Methods] The PilZ domain model was downloaded from the Pfam database. HMMScan software was adopted to scan the genome of GZDF3 strain. Protein conserved domains were analyzed in Conserved Domain Database (CDD). The product of protein was predicted by protein BLAST. The physical and chemical properties of the protein were determined by online bioinformatics analysis softwares in ExPASy website. The recombinant expression vector of the gene was constructed to obtain recombinant protein. [Results] Five genes coding protein containing PilZ domain were discovered in GZDF3 genome. The gene named as Gene4836 encoded a glycosyltransferase by homology analysis. The Gene1423 was a protein-coding gene of YcgR superfamily and Gene1723 encoded hyaluronan synthase belonged to glycosyltransferase superfamily 2. The other two genes Gene2571 and Gene2956 encoded hypothetical proteins. The result of bioinformatics analysis suggested that the molecular weight of the product of Gene4836 was 24.08 kD, and the isoelectric point was 6.39. It was an acidic hydrophilic protein. A conserved PilZ domain was found in the C end of the protein of Gene4836. The optimal expression condition was 0.5 mmol/L lactose for 20 h at 30 °C. A recombinant protein about 25 kD was detected by SDS-PAGE electrophoresis, consistent with the result of bioinformatics analysis. [Conclusion] It is the first report for recombinant expression protein with PilZ domain in Brevibacillus breves. It would provide a foundation for further study of the Pilz domain function.

Key words:Brevibacillus brevis, PilZ domain, glycosyltransferase, prokaryotic expression, recombinant protein