Secreting Expression And Purification Of Ntl, A Lectin From Chinese Narcissus (Narcissus Tazetta Var. Chinensis Roem), In The Yeast Pichia Pastoris

Narcissus tazetta lectin (NTL) is a GNA-like lectin, which exist inChinese Narcisus. Lectins manifest a diversity of activities includingantitumor, immunomodulatory, antifungal, HIV-1reverse transcriptaseinhibitory, and anti-insect activities, which may find practical applications.A small number of lectins demonstrate antibacterial and antinematodeactivities. However, the purification of lectins from raw materials is atime-consuming job and the yield is low. NTL was transformed into E.coli,but there’s lots of inclusion body Therefore, this study is about theexpression of the Chinese narcissus lectin in Pichia pastoris.In this study,the first NTL gene was cloned into pPIC9K-Tvector(constructed in our lab),which was successfully tansformed intoGS115pPIC9K-NTL.SDS-PAGE did not show a significant proteinexpression.Through the rare codons checking,the frequency of use10-20%codons was17, and the frequency of use10%codons was18.Under thehelp of DNA Works and other softwares,a new NTL-opt was synthesized.pPIC9K-NTL-opt was successfully transformed into Pichiapastoris.Using MD medium and PCR,the results showed that every sixtransformants included three positive recombinant Pichia pastoris.Selecting a single clone to express,SDS-PAGE and Western blottingshowed that the protein had been expressed successfully.There’s nosignificant differences between the organic medium and the inorganicmedium expressions.So in the following fermentation,inorganic medium was used.Under the help of Bandscan software,there’s80%targetprotein of the total protein.The characteristic of recombinant NTL protein was compared with thenatural NTL protein.Their agglutination activity were similar: the naturalNTL protein was4.76, recombinant NTL protein was4.39.They hadsimilar secondary structure according to circular dichroism.Thedeglycosylated results showed there’s none.A simple fermentation was carried out in a5L fermenter, at pH5.6,28℃,600rpm. Also, Cells concentration, glycerol concentration andammonium ionsn concentration were studied in the fermentation process.Cells concentration can reach OD600290. Initial glycerol concentrationwas30g/L. At the end of fermentation, the total protein in medium couldreach1000mg/L.