The Tandem Expression Of The Antimicrobial Peptide LfcinB And The Prokaryotic Expression System Of The Myristoylation Product Were Explored

The antimicrobial peptides(AMPs)are members of the immune system producing a kind of important molecules that participate in congenital immune defense after microbial infection.Antimicrobial peptides have a broad spectrum antimicrobial activity and play roles in a special way,making it a kind of potential drugs in the treatment of infection diseases.Some antibacterial peptides have antitumor activities,alone or combined with other traditional medicines shows a good application prospect.To obtain high biological activity of antimicrobial peptides,will promote the development of antimicrobial peptides as progress of anti-infection,anti-tumor drugs.Bovine lactoferricin(LfcinB)is a cationic antimicrobial peptide that exhibits cytotoxic activity against both microorganisms and human cancer cells.We reported here a method of producing tandem multimers of LfcinB using SUMO tag in Escherichia coli.Data showed that expression of the soluble fusion protein with the SUMO tag was more than 90%of total target protein,and dimmer of LfcinB was expressed at the highest level than monomer and trimer when fused with SUMO tag.After using SUMO-specific protease to remove the SUMO tag,and hydroxylamine to release monomer,purified LfcinB was obstained with a yield of 15mg/L.To assay antibacterial and antitumor activity,inhibition zone assay and apoptosis assays were employed.Results demonstrated that recombinant LfcinB exhibited a certain antimicrobial and antitumor activity.These results suggest that the together SUMO fusion system with tandem repeat expression technology provides a potential production method of LfcinB for f-unctional antibacterial peptide studies.Decorated proteins or peptides play an important role in its function.This study discusses the construction of coexpressing of N-myristoyltransferase with CM4 and FUSI in Escherichia coli.(1)Successfully constructing pETDuet-1-CM4 and pETDuet-1-CM4-NMT expression systems,SDS-PAGE suggested that,there are obvious and single band at the corresponding sites.There are substances larger than CM4 in the coexpression complexes by MALDI tandem time-of-flight Mass spectrometric detection.An inhibition zone assay showed that,the purified coexpression complexes displayed higher antibacterial activity to tested S.aureus.Cytotoxicity experiment results showed that,under the same concentration,compared with the CM4,coexpressing products are more likely to cause cell morphology changes of leukemia K562 cells,lead more cells to die.(2)Successfully constructing pETDuet-1-FUS1(MCS1/MCS2).pET28a-FUS1?pSUMO-FUS1?pETDuet-l-FUS1-NMT and pET28a-MetAP co-transformation expression systems,SDS-PAGE suggested that,we only get FUS1 from pSUMO-FUS1 which mostly inclusion.There is little expression from other systems.These results suggest that we have successfully built the coexpression system of NMT and CM4,but it remains to discuss the applicability of this strategy for a variety of different protein or peptide.In conclusion,this study has successfully built the tandem expression system and coexpression systems,got active proteins or peptides,provides references to small molecule peptides or proteins modification for further development of genetic engineering.