Recombinant Expression Of Antimicrobial Peptide Cathelicidin-bf By SUMO Technology In Bacillus Subtilis And Its Biological Function

Antimicrobial peptides (AMPs) are regarded as a potential solution to the worldwide problem of antibiotic resistance among pathogens because of their ability to kill microbes directly and boost specific innate immune responses in the host. Cathelicidin-BF (CBF), the first identified cathelicidin antimicrobial peptide in reptiles, has been purified from the venom of the banded krait snake species Bungarus fasciatus. This peptide has potent antimicrobial activities against many microorganisms. In the present study, we aimed to establish an efficient method for producing recombinant CBF using SUMO technology in a B. subtilis expression system.1. Expression and purification of fusion protein SUMO-CBFIPTG (0.25mM,0.5mM,1mM and2mM), Incubation temperatures (16℃,25℃,30℃and37℃), induction time (0-18h) were examined in an attempt to maximize extracellular production of SUMO-CBF. We found that IPTG,30℃and12h was the optimal induction condition. The amount of SUMO-CBF in whole cells and in the supernatant at12h was examined by western blot, and the majority of the produced fusion protein was secreted into the medium. SUMO-CBF secreted from the WB800N/pHT43/his-SUMO-CBF culture supernatant was concentrated by ammonium sulfate precipitation. Fusion protein SUMO-CBF was then purified to near homogeneity by affinity and anion exchange chromatography. A yield of～22mg SUMO-CBF was obtained per liter of culture medium. The purity was>90%, based on densitometry using Quantity One software.2. Expression and purification of SUMO protease1To maximize the activity of extracellular SUMO protease1, different incubation temperatures were also examined. Western blotting analysis was used to determine the extracellular activity by detecting the amount of SUMO protease1, SUMO-CBF, and cleaved SUMO. Results indicated that SUMO protease1secreted in LB medium by WB800N/pHT43/his-SUMO protease1was highly active. We determined that30℃was the optimal temperature for the expression and secretion of SUMO protease1. Western blot analysis indicated that SUMO protease1activity in the culture medium increased during the0-10h interval and then decreased. Western blotting analysis also confirmed that the majority of the SUMO protease1was secreted into the medium. One liter of recombinant WB800N/pHT43-SUMO protease1culture supernatant was then dialyzed overnight against a low salt concentration buffer, and SUMO protease1was purified by cation exchange and affinity chromatography. A yield of～1mg SUMO protease1, with a purity of90%, was obtained from11of culture medium. 3. Enzymatic cleavage of SUMO-CBF fusion protein and purification, characterization of recombinant CBFFollowing incubation at4℃for12h, the reaction mixtures were analyzed by SDS-PAGE. Results indicated that0.05μg of the purified enzyme was sufficient to achieve80%cleavage of2.5μg of the fusion protein, giving an enzyme-to-substrate ratio of1:50. Together, these results indicated that the activity of SUMO protease1purified from B. subtilis was slightly lower than that of SUMO protease1purified from E. coli.The cleavage reaction mixture was reapplied to a Ni-NTA resin column. Recombinant CBF was purified in the pass-through fraction with80%homogeneity. Following dialysis against a low salt concentration buffer, recombinant CBF was further subjected to a Resource15S cation exchange column. The purity of the resultant recombinant CBF was>95%, as detected by densitometry. A final yield of～3mg recombinant CBF was achieved per liter of bacterial culture.Recombinant CBF displayed similar activity to that of synthetic CBF, resulting in an MIC of1μg/ml for E. coli ATCC25922,4μg/ml for E. coli K88,2μg/ml for E. coli K12,4μg/ml for P. aeruginosa CMCC27853,4μg/ml for S. aureus ATCC25923.4. Effect of recombinant CBF on protection against E.coli K88infection in miceFor this experiment, an intravenous (iv) model of E.coli K88infection in mice was established.In the aspect of gut microbial population, the administration of recombinant CBF before infection significantly reduced E.coli population and the percentage of E. coli in the total bacteria population compared with the challenged counterpart, and ifidobacterium and Lactobacillus population and the percentage of E. coli in the total bacteria population were significantly increased.In the aspect of intestinal morphology, mice tended to exhibit greater villous atrophy and crypt depth reduction in the intestinum jejunum after challenge. Challenged mice showed significantly reduced villous height and villi/crypt ratio compared with the non-challenged counterpart. The administration of recombinant CBF before infection significantly increased villous height and villi/crypt ratio compared with challenged mice. In the aspect of the expression of tight junction proteins, challenged mice showed significantly decreased expression of claudin-1, ZO-1and ZO-2(p<0.05) compared with the non-challenged counterpart. The administration of recombinant CBF before infection significantly increased the expression of claudin-1, occludin, ZO-1and ZO-2(p<0.05) compared with the challenged counterpart. In the aspect of the serum immunoglobulin, challenged mice showed significantly higher serum IgA, IgG and IgM level compared with the non-challenged counterpart. The administration of recombinant CBF before infection significantly reduced IgA, IgG and IgM level in serum compared with the challenged counterpart. In the asepct of the level of intestinal sIgA, challenged mice showed significantly higher sIgA level compared with the non-challenged counterpart. The administration of recombinant CBF before infection significantly reduced sIgA level compared with the challenged counterpart. In the asepct of the rate of spleen lymphocyte transformation, challenged mice showed significantly higher rate of spleen lymphocyte transformation compared with the non-challenged counterpart. The administration of recombinant CBF before infection significantly reduced the rate of spleen lymphocyte transformation compared with the challenged counterpart. In the asepct of the expression levels of intesitnal cytokines, including TNF-α,MCP-1and IL-10were significantly increased. The administration of recombinant CBF before infection significantly reduced the expression levels of TNF-α,MCP-1and IL-10.