ELP-intein Mediated Expression And Purification Of Human Beta Defensin-3

ObjectiveAntimicrobial peptides (AMPs) which are secreted by most living organisms haveshown robust activity against a wide variety of pathogens, including drug-resistantbacteria. They are important components of innate and adaptive system. AMPs areknown to exist in all kingdoms. So far, more than1700types of peptides withantimicrobial activity have isolated from various cells and tissues. AMPs affectmicroorganisms through membrane or target cell contents disruption as well asselective killing as part of the primary or secondary mechanism of action. AMPs haveseveral advantages over antibiotics in attractive antimicrobial activity part. Therefore,it is of great importance and necessity to do deep researches for antimicrobial peptides.Human beta-defensin-3is an endogenous, cysteine-rich antimicrobial peptide thatresistants to high salt and contributes more to host defense against multi-resistantbacteria compared with other beta-defensins. In present, the main methods of yieldinghuman beta-defensin-3are chemical synthesis and recombinant DNA technology. Itcosts a lot and spares more time by the former method. However, it is also not an easy way to improve yields by engineering expression. There is a great need for a lessexpensive and efficiency peptide-production platform.This research aims atexpressing human beta-defensin-3in E.coli BLR(DE3) via pET-EI plasmid vectorwhich developed by Dr. Davaid. It will lower cost as well as establish the foundationto large-scale production of HβD-3.Methods1Based on E. coli codon preference, transformed into two novel sequences namedHβD-3i and HβD-3ii respectively by DNA club software.2Adopted the method of PCR-based gene SOEing to synthesize HβD-3/HβD-3i/HβD-3ii DNA sequences artificially.3Constructed expression vector pET-EI-HβD-3/HβD-3i/HβD-3ii following thesteps of double digestion and connecting reaction, then transfered the vectorpET-EI-HβD-3/HβD-3i/HβD-3ii to E.coli Competent Cell JM109, the positive clonewould be identified by sequencing.4Prepared E.coli Competent Cell BLR(DE3), then transfered correct positive clonesinto BLR(DE3).Identification was engaged by PCR positive plasmid vector.5Preliminary attempted to express positive recombinant transformant in smallquantity.6Optimization of expression condition in order to figure out the most appropriatetemperature and time.7Recombinant expressed in the condition of the most appropriate temperature andtime in medium quantity.8Utilized the centrifugation of “Inverse Transition Cycling, ITC” to purify fusionprotein. Optimization of ITC condition. Using Tricine-SDS-PAGE which is suitableto small molecular protein to testify target protein. Then concentrating target protein.Using BCA protein kit to detect target protein concentration.9Preliminary attempted to testify the antimicrobial activity of recombinant peptidesby agarose diffusion experimental on Salmonella and Staphylococus aureus. Utilizingminimum inhibitory concentration to quantitative testify the antimicrobial activity of recombiant peptide on Salmonella and Staphylococus aureus. By analysing thegrowth curve of Salmonella and Staphylococus aureus after killing bacteria to processkinetic study of recombinant peptides.Results1Constructed expression vector pET-EI-HβD-3and pET-EI-HβD-3i/HβD-3ii thatbased on E. coli codon preference successfully.2Preliminary attempted to express positive recombinant transformant in smallquantity, and figured out that the most appropriate expression temperature was12~14℃,and expression for48h was recommended for higher yields. The cleavingreaction could be more quickly at37℃,and timing of cleaving time-course sampleswas better to stop after24h.3The final yields of HβD-3/HβD-3i/HβD-3ii was respectively0.123mg/mL,1.479mg/mL,1.359mg/mL under the most appropriate temperature and timingcondition.4Results showed that bacteriostatic annulus of HβD-3/HβD-3i/HβD-3ii whoseconcentration was120ug/mL on Staphylococus aureus and Salmonella were clearwhich had no different with kanamycin.While HβD-3/HβD-3i/HβD-3ii whoseconcentration was2ug/mL exhibited no antimicrobial activity.5The MIC of HβD-3/HβD-3i/HβD-3ii on Salmonella and Staphylococus aureuswere8ug/mL、4ug/mL、4ug/mL，4ug/mL、4ug/mL、2ug/mL respectively.6By analysing the growth curve of Salmonella and Staphylococus aureus afterkilling bacteria to process kinetic study of recombinant peptides, the results showedantimicrobial peptides inhibited the growth of Salmonella and Staphylococusaureus.ConclusionThe HβD-3/HβD-3i/HβD-3ii genes were cloned into a plasmid vector pET-EIcontaining the tags elastin-like peptide(ELP) and intein to construct the expressionvector pET-EI-HβD-3/HβD-3i/HβD-3ii. HβD-3i/HβD-3ii were12times higher yields than HβD-3. All the peptides, expressed as fusion protein, were isolated fromthe protein debris by the method called Inverse Transition Cycling(ITC).Fully reducedpeptides that were purified exhibited expected antimicrobial activity that towarded toStaphylococus aureus was stronger than Salmonella.It is an attractive way to expressHβD-3/HβD-3i/HβD-3ii via pET-EI prokaryotic plasmid vector. This approachdescribed here is a low-cost, convenient and potential way for large-scale humanbeta-defensin-3production.