Soluble Expression And Characterization Of Pinellia Ternata Agglutinin In Engineering Bacteria

Plant lectins are non-immune origin and carbohydrate-binding proteins thatwidely exist in dicotyledonous and monocotyledonous plants. These proteins arecalled lectins for their functions in recognizing and binding to specificpolysaccharides or glycoproteins on cell surfaces. It is generally believed that lectinscan trigger several important cellular processes. Furthermore, plant lectins as potentialantineoplastic drug have been applied in bench scale and clinical trials. In the past fewyears, numerous plant lectins were purified and characterized in detail to elucidatevarious characteristics and functions.Pinellia ternata agglutinin (PTA), a member of monocot mannose-binding lectinsand a homodimer that consistes of two N-terminus domains (P-D1) and twoC-terminus domains (P-D2), having antitumor potential from the tubers of P. ternata. P.ternata, which belongs to Pinellia, Araceae species, is widely used in China as animportant traditional Chinese medicinal herb. Bio-activity assays of PTA also suggestsits potential application as antineoplastic drugs. Since Yao firstly obtained thefull-length cDNA of PTA, the recombinant protein encoded by PTA gene wassuccessfully expressed in Escherichia coli and Bombyx mori cell. But disadvantagessuch as insolubleness or unsuitability for large-scale culture are unavoidable forprevious strategies.Human SUMO fusion systems and Pichia pastoris expression systems were usedto express P-D2and P-D1, respectively. The plasmid pET-32-SUMO-P-D2andpPIC9k-P-D1had been constructed correctly before they were transformedrespectively into strains BL21(DE3) and SMD1168. The positive strains wereinduced by IPTG or methanol and recombinant protein was purified by Ni–NTAchromatograpHy. About12mg/L of purified P-D1was obtained and the yield of P-D2was about5mg/L.The result of inhibiting fungal revealed P-D1has potent inhibition againstAlternaria altanata at low concentration. According to the carbohydrate inhibition assay, P-D1and P-D2have the capacity of hemagglutination on mice erythrocytes atthe concentration of6μg/mL, which could be inhibited by specific sugars, such asD-mannan, excluding D-lactose and D-glucose. P-D1and P-D2inhibit obviously thegrowth of tumor cell lines (SW-620, Bel-7404) in a dose-dependent pattern. P-D1alsoinhibits the growth of tumor cell line SMMC7721. Inhibition ratios of P-D1andnative PTA were relative to time and concentration in Bel-7404. The inhibition curvesof native-PTA and P-D1are similar, both of which are steeper at the beginning andthen become relatively flat with increased concentration. Those lectin proteins alsoinduce apoptosis of Bel-7404. The longer time and the higher concentration, the moreobvious of apoptosis pHenomenon. But mechanism of lectin protein inducingapoptosis will need further investigation.This paper also reports that the recombinant protein product, similar to nativePTA purifed from tubers of P. ternate, possesses biological functions. In this work,lectin protein was solubly expressed in human SUMO fusion system and P. pastorisexpression system for the first time, suggesting potential application to the expressionof other plant lectins.