Screening Of The Substrate Of Site-2 Protease Like Protein From Rhodococcus Ruber And Its Fusion Expression And Functional Characterization

Site-2 protease(S2P),a kind of metalloprotease present in most bacteria,plays an important role in regulated intramembrane proteolysis.What is the substrate of S2P like protein from Rhodococcus ruber is not yet known.The signal transduction pathway involved in its substrate remains elusive.Screening of the substrate of S2P like protein from Rhodococcus ruber SD3 and functional analysis was conducted in the study,which paved the way for unveiling the signal transduction pathway involved in regulated intramembrane proteolysis in Rhodococcus ruber SD3 and provided potential targets for genetic modification of Rhodococcus ruber SD3.The main research contents and results of this paper are as follows:Firstly,the substrate of site-2 protease like protein from Rhodococcus ruber SD3was screened using molecular docking in the present study.The gene of substrate protein was cloned and its bioinformatics analysis was performed.A substrate of site-2 protease was found to be histidine kinase(RHK)after screening.The sequence accession number of the gene in Gen Bank was MN688330.Its open reading frame was 1410 bp in length and encoded 469 amino acids.The theoretical molecular weight of the protein was 49.62 k D.The probability of signal peptide existence was0.6765 and the probability of signal peptide anchoring was 0.2113.The cleavage site of signal peptide was located between amino acid residue 35-36.It belonged to unstable hydrophobic protein with three obvious hydrophobic peaks.It had no disulfide bond and contained one transmembrane region.The secondary structure was mainly?-helix.The subcellular localization indicated that histidine kinase played a biological role on cell membrane.A variety of conserved domains in histidine kinase were contained between amino acid residues 164 and 455,which belonged to various superfamilies.The tertiary structure was built using Robetta modeling tool.Phylogenetic tree analysis showed that histidine kinase from Rhodococcus ruber SD3and Rhodococcus pyridinivorans SB3094 gathered into a cluster.Fluorescence quantitative PCR analysis indicated that the expression of histidine kinase decreased to 0.76 folds and 0.78 folds under toluene and phenol stress as compares with control,respectively.Secondly,catalytic activity and kinetics of histidine kinase fusion protein were studied.Sequence analysis of rhk gene revealed that it had more rare codons,high GC content and some repetitive sequences.Therefore,rhk gene of Rhodococcus ruber SD3 was codon-optimized,and the optimized gene was named after rhks,then recombinant expression plasmid p GEX-4T-2-rhks was constructed.The plasmid was transformed into E.coli BL21(DE3)for heterologous expression,and the expression conditions were optimized.At the induction conditions of 25°C and 1 m M IPTG,RHK fusion protein was successfully expressed and had catalytic activity.GST-RHK fusion protein with electrophoresis purity was obtained by glutathione agarose affinity chromatography,with purification folds of 3.1 and recovery rates of 19.5%.The protein was estimated to be 72.75 k D,approximately in accordance with the theoretical value of 72.17 k D.The recombinant protein was further confirmed by LC-MS/MS analysis.Kinetics analysis indicated that Km?Vmax and Kcat were 20.92?M?0.17?M/min and 1.4 min^-1,respectively.Thirdly,to study the relationship of rhk and organic solvents tolerance of Rhodococcus ruber,the gene enhancement and gene knockdown of rhk in Rhodococcus ruber SD3 were performed to obtain rhk gene enhancement strain(sdrhk E)and gene knockdown strain(sdrhk D).The changes of RHK expression of strain sdrhk E and strain sdrhk D were studied using q PCR.The results showed that the RHK expression of strain sdrhk E was up-regulated to 46.57 folds as compared with the control sample and the RHK expression of strain sdrhk D was down-regulated to0.05 folds.The growth of strain sdrhk D was better than wild type,whereas the growth of strain sdrhk E was less inferior than wild type when cultured in the medium containing phenol,toluene,chlorobenzene and isooctane,respectively.The results suggested that the down-regulation of rhk gene may increase the organic solvents tolerance of strain.However,the up-regulation of rhk gene may decrease the organic solvents tolerance of strain.In conclusion,one kind of substrate of S2P like protein was found to be histidine kinase using molecular docking screening.The histidine kinase fusion protein with catalytic activity was obtained by heterologous expression.The relationship between histidine kinase and organic solvents tolerance of Rhodococcus ruber was demonstrated.The study paves the way for unveiling the relationship between signal transduction pathway involved in regulated intramembrane proteolysis and organic solvents tolerance in Rhodococcus ruber SD3.