Expression, Purification And Antifungal Characterization Of Pinellia Ternate Agglutinin

Lectins are naturally occurring proteins and glycoproteins which bind selectively and non-covalently to carbohydrate residues.They are widely distributed in living organisms including animals,plants,microorganism.The main characteristic of this class of protein is their ability to interact specifically with carbohydrates and to combine with glyco-components of the cell surface.On the basis of recent advances in biochemistry and molecular cloning and structural analysis,virtually all currently known plant lectins can be classified into seven families according to structurally and evolutionarily-related proteins: amaranthins,Cucurbitaceae phloem lectins,lectins with hevein domain(s), jacalinrelated lectins,legume lectins,monocot mannose-binding lectins and type-2 ribosome-inactivating proteins.Pinellia ternata is a traditional Chinese medicinal plant species,belonging to the family Araceae.Araceous lectins are a group of proteins possessing at least one non-catalytic domain that binds reversibly to specific monoor oligo-saccharide(glycoconjugate).They readily agglutinated rabbit erythrocytes but were unable to agglutinate human erythrocytes irrespective of the blood group.The property of Pinellia ternate angustate agglutinin(PTA) and its domains was investigate in this study.The pta cDNA sequence encoding for mature PTA protein and two domains were cloned into PET-28a plasmid and the resulting in pET-28a-PTA,pET-28a-DOM1 and pET-28a-DOM2 containing N-terminal His-tagged fusion protein,repectiveLy.The expressions of them in E.coli BL21 were investigated.Purification of the PTA-P,PTA-DOM1 and PTA-DOM2 recombinant proteins were performed under native conditions by nickel ion metal-affinity chromatography.The recombinant PTA-P,PTA-DOM1 and PTA-DOM2 exhibited several characteristics:1.they exhibited hemagglutinating activity towards rabbit but not human erythrocytes,and their hemagglutinating activity could be inhibited by mannose.These profiles were similar to other Araceae lectins.2.Both PTA-P and two domains exhibited potent antitungal activity toward Alternaria altanata,Bipolaris sorokiniana and Curvularia lunata.The antifungal activities of PTA-P were obviously stronger than that of PTA-DOM1 and PTA-DOM2.Among the tested fungi, Alternaria altanata appeared the most sensitive to PTA-P with an IC50 of 0.19μM, Alternaria altanata and Bipolaris sorokiniana more sensitive than Curvularia lunata to PTA,PTA-DOM1 and PTA-DOM2.The morphological toxicity of PTA-P,PTA-DOM1 and PTA-DOM1 to Curvularia lunata was revealed by an increase in mycelial apex offshoot and tumescence after treatment with 50ug proteins for 24 h.3. The results of thermal denaturation of fusion proteins showed that the antifungal activity of PTA-P,PTA-DOM1 and PTA-DOM1 were extremely stable between 20℃and 60℃.Even heating at 100℃for 5 min caused loss of only 25%of its original activities.4.they also exhibited activity under a broad pH range from 4.0 to 9.0 with highest activity at 7.8.We also found that the three proteins were active against Escherichia coli and Stapylococcus aureus.Sum up,The PTA-P,PTA-DOM1 and PTA-DOM1 may be a promising candidate for possible applications in the genetic engineering of disease-resistant crops.