Purification, Expression And Bio Activity Analysis Of The Proteins From Pinellia Ternata

Pinellia ternata (Thunb.) Breit belongs to Araceae family, Pinellia. The tubers ofP. ternata, a traditional Chinese medicine herb is widely consumed in ancient China.containing protein, polysaccharide, alkaloid and orgainic acid. The tubers areconsumed to stop coughing, phlogistic, nausea and vomiting. Reseaches indicate P.ternata has many biofunctions including termination of pregnancy, anti-tumor,anti-pest, anti-virus and anti-fungal, anti-arrhythmia, promoting of motominic,reducing blood fat and agglutining hemocytes. The total protein of P. ternata includesPinellia ternata tryspin inhibitor (PTTI) and P. ternata agglutinin (PTA).To date, plant tryspin inhibitors and lectins have been widely used in commercialapplications due to the bioactivities. In this study, we established a novel single-stepTrypsin-Sepharose4B affinity purification method for the isolation of PTTI. Thenovel plant inhibitor was obtained after homogenization, extraction, ammoniumsulfate precipitation gradient, affinity binding and elution. SDS-PAGE indicated PTTIappeared as a single band of about20kDa, which indicating PTTI belonged to Kunitztype plant protease inhibitors. Trypsin inhibitor activity of purified PTTI wasmeasured using BAPNA as substrate and porcine pancreas trypsin as source ofenzyme. The specific inhibitory activity of purified PTTI was calculated to be366.5±14.57units per mg protein. The calculated purification fold of PTTI by the specificinhibitory activity was about8.6. The polysaccharide content analysis indicated PTTIwas a glycoprotein with a degree of12.82±0.64%polysaccharide. The thermal andpH stability assay indicated the trypsin inhibitory activity of PTTI was stable up totemperature and pH.Pinellia ternata agglutinin, a monocot mannose-binding lectin, is a homologusdimer composed of four subunits. PTA was high-throughput isolated by optimzingpurification condition using Mannose-Sepharose4B affinity chromatography for thefirst time in this work. The total protein of tubers of P. ternata was obtained afterhomogenization, extraction, ammonium sulfate precipitation, and dissolved in different concentrations of NaCl solution ranging from10%-30%to activate itsbinding affinity toward the column. PTA was bound to the affinity column by loadingof the total protein into the column and elution using PBS buffer. The maximumpurification yield (35.5mg/g) was obtained when PTA was treated with25%(w/v)NaCl solution, and the molecular weight of PTA analyzed by SDS-PAGE was12kDawith a purity of~97%. The agglutination property of purified PTA was confirmed bymouse erythrocytes, which indicated PTA agglutinated obviously mouse erythrocytesat6μg/mL. A novel single-step Mannose-Sepharose4B affinity chromatographymethod was established. The proposed method has great potential for industrialapplication because of its advantages, which include rapid isolation, high purity, highyield, low cost, and minimal requirement of chemical materials compared to ionexchange chromatography, gel chromatography and Aasialofetuin-Sepharose4Bchromatography.The target peptide can be targeted into periplasm depending on the secretionfunction of alkaline phosphatase signal peptide (APSP). After translocation, the signalpeptide is specifically recognized and automatically cleaved by a membrane-boundsignal peptidase. Disulfide bonds and proper folding are formed by a series ofenzymes. The pta-n gene encoding N-terminus domain was cloned by PCR usingPCR primer according the structure information of PTA. The fused gene apsp-pta-nwas obtained by3-step PCR according to the nucleotide sequence of apsp. The fusednucleotide sequence apsp-pta-n was inserted into pET-28a prokaryotic expressionvector by restriction enzyme digest sites (Nco Ⅰ and Xho Ⅰ), and thenoverexpressed in E. coli BL21(DE3) cells by IPTG induction. Ni-NTAchromatography was used for the purification and about20mg/L purified PTA-N wasobtained. SDS-PAGE indicated PTA-N was expressed solubly and purified byNi-NTA chromatography. Hemagglutination assay indicated PTA-N could agglutinatemice erythrocytes. anti-fungal activity against Gibberella saubinetii was demonstratedand the in vitro anti-proliferative activity towards human tumor cell lines wasconfirmed by MTT method. Further Hochest33342nuclear staining and DNAfragmentation assay of Bel-7404showed typical apoptotic body and nucleosomal DNA fragmentation. These results indicated PTA-N possessed several bioactivities,similar to native PTA. The plant mannose-binding lectin was expressed solubly inprokaryotic expression system for the first time by fusing alkaline phosphatase signalpeptide with the lectin protein according to strucutre information of PTA, providing anew strategy for plant lectin expression.