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

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.