The Construction And Prokaryotic Expression Of PTWIN1-Lactoferricin B And PTWIN1-BNBD5

Antimicrobial peptide (AMP) is a kind of small molecular polypeptide less than100amino acids residue, with a specific biological activity, which is innated defense substance oforganism with long-term evolution. It could risist Gram-positive and negative bacteria, fungi,protozoa, viruses and tumor cells but having no damage to normal cells.Bovine lactoferrin (Lactoferricin B, LacB) is located between17th and41th amino acidof bovine lactoferrin, composing of25amino acids, with amphiphilic β-fold molecularstructure. LacB has wide antimicrobial spectrum, and strong antibacterial and sterilizationeffect. However, the lack of source LacB hindered its application.Cows endometrial epithelial cells AMP (BNBD5) is a45amino acid polypeptide of theβ-defensins. However, cows produce very little BNBD5. Moreover, synthetic BNBD5peptides generally have no active.In recent years, with the rapid development of genetic engineering techniques,purification of the protein also has a new breakthrough. IMPACT-TWIN system with self-splicing intein is selected instead of the traditional protease strategy, which simplifypurification steps and reduce costs. In addition, two prokaryotic expression vectors pTWIN1and pTWIN2of the system allow efficient expression of foreign gene in Escherichia coli,facilitating collection and purification of expressed proteins.Ternary fusion expression plasmid could be constructed when the target gene was clonedinto vector pTWIN1or pTWIN2, which contains corresponding nucleic acid sequence ofchitin-binding domain (CBD) and intein. When the fusion protein flowed through the chitinresin column, CBD could be adsorbed on the resin. With changing PH, temperature, or addinga reducing agent, the peptide bond between intein and target protein cleaved. The targetprotein is eluted, because the CBD and intein polypeptide was still bound to the resin. Thetarget protein can be purificated effectly.In this study, overlapping PCR technology was applied to obtain the complete LacBsequence with two complementary primers. Then the pTWIN1-Lactoferricin B wasconstructed after the LacB PCR products were inserted into prokaryotic expression vectorpTWIN1. At the same time, the whole gene of BNBD5was synthesised and cloned into pTWIN1, so pTWIN1-BNBD5was obtained. The two prokaryotic expression vectors thenwere transformed into E. coli BL21and the recombinant fusion proteins were induced tosuperexpress and the Lac B and BNBD5were attempted to be purified.The results showed that, target gene LacB and BNBD5were obtained successfully, andalso were correctly inserted into prokaryotic expression vector pTWIN1carrying the CBDand intein. The fusion polypeptide with target protein was induced to express in the form ofinclusion bodies in E. coli, which is undissolving in common buffer and difficult to bepurified. The denaturing and refolding experiments of the including bodies were carried out.LacB could be partially dissolved, but not smoothly purified, while BNBD5remainsinsoluble.Therefore, the expression and purification of intein-mediated LacB and BNBD5with thepTWIN1as expression vector was facing problems. In future research study should focus onsolubility and activity of inclusion. In addition, it is necessary to research whether LacB andBNBD5are fit for the IMPACT purification systems.