Multimerization, Expression Of Plectasin Gene From Pseudoplectania Nigrella In Escherichia Coli And Bioactivity Analysis Of The Recombinant Products

To improve the antimicrobial activity of plectasin, the sequence coding Pseudoplectania nigrella plectasin was optimized and the multimeric genes of plectasin were constructed. Both genes were successfully expressed in Escherichia coli, and the antimicrobial activity and hemolysis of the recombinant products were characterized. The main results were listed as following:1. Expression of the plectasin monomer gene in E. coliAccording to the codon usage bias of E.coli, the Ple(pe) gene coding the Pseudoplectania nigrella plectasin was amplified by SOE-PCR based on the amino acid sequences. The nucleotide sequence (140bp) of the optimized Ple(pe) gene was identical to the native gene. The optimized Ple(pe) gene was cloned into plasmids pGEX-4T-1and pET-30a(+), and successfully expressed in E.coli BL21(DE3). The molecular weight of the recombinant Ple(pe) fused with GST-tag and His-tag were estimated by SDS-PAGE to be31kDa and11kDa, respectively. The GST-Ple(pe) protein was6folds greater than the His-Ple(pe) protein. The amount of GST-Ple(pe) protein was increased by40%when the plasmid pGEX-Ple(pe) was transformed into E.coli BL21(DE3)pLysS.2. Construction and expression of tandom multimers of plectasin gene in E. coliTo amplify the plectasin monomer gene, namely Ple(pes), PCR was firstly performed using Ple(pe) gene as template. The Ple(pes) gene containing XhoI and SaiI site which generated identical cohesive ends was designed for the construction of plectasin multimers. Using the co-adhesive end restriction and ligation strategy, the dimer, trimer, tetramer and octamer of plectasin were multimerized by T4DNA ligase. The multimeric genes of plectasin were successfully inserted into plasmids pGEX-4T-1. The resulting vectors were introduced into E.coli BL21(DE3)pLysS for recombinant protein expression. After IPTG induction, the dimmer to tetramer except octamer were successfully expressed to be36kDa,41kDa and46kDa, respectively. After IPTG induction for4h, the expression levels of the fusion proteins were significantly increased, which accounted for35.7%,26.9%and12.1%of total cell proteins, respectively. In addition, the tetramer continued to increase significantly at1～4h induction time, and remained steady over the range of time induction.To analyse the antimicrobial activity in vivo, the recombinant plasmids pGEX-Ple(pe), pGEX-Ple(pes-2) and pGEX-Ple(pes-3) were transformed into E.coli DH5a, respectively. After IPTG induction, the growth of host cells was monitored by measuring the optical density at OD6oonm.The efficient time of the recombinant products against host cells was1～3h,1～5h and1～7h post-induction respectively. Meanwhile, after induction for2h, the growth inhibition of host cells was most significant. The results of cell growth demonstrated that all of the recombinant products definitely inhibited E.coli DH5a. With the increase of plectasin gene number, antimicrobial effects of recombinant products significantly increased and the antimicrobial time relatively extended.3. Purification, cleavage and bioactivity of the recombinant plectasinThe induced cells were lysed and recombinant products were purified. Results showed that68.9%of the GST-Ple(pe) was in soluble form. The expression levels of the fusion proteins GST-Ple(pes-2) and GST-Ple(pes-3) accounted for35.7%and26.9%of total cell proteins respectively. After GSTrap FF purification, the purity quotient of fusion proteins were above90%. The amounts of purified fusion proteins GST-Ple(pe), GST-Ple(pes-2) and GST-Ple(pes-3) were1269.3μg/mL,365.5μg/mL and107.6μg/mL, respectively. The GST-Ple(pe) and GST-Ple(pes-2) were effectively cleaved by thrombin. The yields of the dialyzed Ple(pe) and Ple(pes-2) with the purity quotient of95%reached296.4μg/mL and138.5μg/mL, respectively.A modified broth microdilution assay was used to determine the antimicrobial activities of Ple(pe) and Ple(pes-2).The recombinant products showed strong activity against several species of Gram-positive bacteria including S.aureus ATCC25923, S.pneumoniae ATCC49619, S.pyogenes ATCC19615and S.suis CVCC3309, but showed no or limited activities against E.faecalis ATCC29212, B.subtilis ATCC6633, E.coli ATCC25922and C.albicans ATCC10231. The antimicrobial activities of Ple(pe) and Ple(pes-2) were similar to the synthesized plectasin, and the antimicrobial activity of the Ple(pes-2) was higher than the Ple(pe). Both the Ple(pe) and Ple(pes-2) had no hemolytic activity for porcine erythrocytes.