The Expression And Bactericidal Activity Of Recombinant Regenerating Protein3

The gastrointestinal tracts of mammals are heavily colonized withvast symbiotic microbial communities. Maintaining the mutuallybeneficial nature of this relationship requires strict sequestration ofresident bacteria in the intestinal lumen, as microbial incursions acrossepithelia can elicit inflammation and sepsis. Epithelial antimicrobialproteins are key elements of intestinal mucosal defense, they play animportant role in maintaining mutually beneficial host-microbialrelationships.The Reg3(regenerating protein3) proteins belong to the epithelialantimicrobial proteins. As we described previously, expression of murineReg3γ mRNA was enhanced in mouse intestinal mucosa in bothbacteria-reconstituted and dextran sulfate sodium (DSS)-induced colitis.The expression of Reg3A mRNA was also up-regulated in the colonicepithelial cells in inflammatory bowel disease (IBD). Hence, Reg3proteins are thought to contribute substantially to the bacteria-hostinteraction in the gut.An increasing number of studies provide evidence of participation ofReg3proteins in the proliferation and differentiation of diverse cell typesas anti-inflammatory, antiapoptotic and mitogenic agents. Moreover,Reg3family members are associated with various pathologies, includingdiabetes and forms of gastrointestinal cancer. However, the regulationand the antimicrobial mechanisms for these portein are still not completely understood. It is necessary to establish an efficient expressionand purification method of recombinant Reg3proteins to further analysisof Reg3A proteins.In this study, murine Reg3γand human Reg3A amplicon wasgenerated by PCR. PCR products were inserted into pET30b (+) vectorand transformed into DH5α competent cells. Purified plasmids were thentransformed into E. coli BL21-CodonPlus (DE3)-RIL for the expressionof recombinant proteins.Expression strains were grown at37°C in500ml of LB Broth for4h.Recombinant Reg3proteins were expressed with the induction byIPTG(0.5mM,6h). The cells were ruptured by sonication. Inclusion bodythat containing the recombinat protein were homogenized andresuspended in Resuspension Buffer. The re-suspended inclusion bodieswere added to250ml of Refolding Buffer in a drop-wise manner. After24h incubation at the room temparture, the insoluble material wasremoved by centrifugation, and dialyzed in Dialysis Buffer1overnight,followed by a second overnight dialysis in Dialysis Buffer2. Thedialysate was centrifuged at10,000g for30min, and the supernatant wasfilterized and purified passed over ion-exchange column and gel filtrationcolumn.To further confirm the recombinant Reg3proteins identities, thepurified proteins were analyzed by SDS-PAGE and followed by Westernblotting. Reg3proteins resulted in single band at~16kD, corresponded tothe predicted molecular mass of the mature proteins (16.5kD).Reg3proteins can be cleavage by trypsin resulting in a lowermolecular (~15kD) form and an11-amino acid fragment. To test whetherthe recombinant proteins have the same characteristics, we performedtrypsin digestion to recombinant Reg3γ and Reg3A. we demonstrated the incubation of recombinant Reg3γ and Reg3A with trypsin for1h at37℃yielded the formation of a lower molecular weight band at~15kD.The trypsin-excised products of recombinant proteins were analyzedfor its solubility. Purified recombinant proteins (Reg3γ and Reg3A) weredigested with the trypsin. The reaction samples were centrifuged for30min at20,000g at4℃. The supernatants were recovered and theresulting pellets were resuspended in1ml of the buffer and centrifugedagain. The second supernatant was discarded. Both the first supernatantsand final pellets were analyzed by SDS-PAGE and Coomassie bluestaining. We showed that the trypsin-excised type of Reg3γ and Reg3Aproteins displayed the relative distribution in the pellet fraction, whereasReg3γ and Reg3A was in the supernatant faction.To confirm whether the recombinant proteins have bactericidalactivity as natural ones, we added purified recombinant Reg3γ andReg3A proteins and their trypsin-excised products to Enterococcusfaecalis, a Gram-positive bacteria, and observed a dose-dependentreduction in the viability of bacteria in CFU assay. Trypsin-digestedrecombinant Reg3γ and Reg3A exhibited enhanced antibacterial activitycompared of uncleaved recombinant proteins. However, the recombinantproteins and trypsin-digested ones didn’t exhibit any bactericidal activityto Gram-negtive bacteria Escherichia coli.Together, our findings will lay a foundation for further detailedanalyses of Reg3proteins functions.