title: Expression and purification of the trypsin inhibitor from tartary buckwheat in Pichia pastoris and its novel toxic effect on Mamestra brassicae larvae.
abstract: The gene of the trypsin inhibitor of tartary buckwheat (Fagopyrum tataricum) was successfully cloned, expressed in Pichia pastoris and tested for regulatory effects on insect growth. The three significant factors were optimized by single-factor experiments and central composite design in response surface methodology. Proteins were efficiently expressed at levels of 489.6-527.4 U/mg in shaken flasks. The trypsin inhibitor from tartary buckwheat (FtTI) was purified by affinity chromatography and centrifugal ultrafiltration. The purified FtTI efficiently inhibited trypsin protease activity by competitive inhibition with a Ki value 1.5 nM. The molecular mass of the purified protein was approximately 13.8 kDa. FtTI had a higher toxic killing effect on Mamestra brassicae larvae. The median lethal concentration for the larvae was 15 μg/mL.
authors.affiliation: College of Life Sciences, Sichuan Agriculture University, Ya'an, 625014, Sichuan, People's Republic of China, ruanj741@yahoo.com.cn.
authors.forename: Jingjun
authors.initials: J
authors.lastname: Ruan
authors.forename: Jun
authors.initials: J
authors.lastname: Yan
authors.forename: Shengqi
authors.initials: S
authors.lastname: Hou
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
authors.forename: Xueyi
authors.initials: X
authors.lastname: Han
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D019612:Fagopyrum / Q000737:chemistry*; D007814:Larva / Q000187:drug effects / Q000254:growth & development; D009036:Moths / Q000187:drug effects*; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000633:toxicity; D012044:Regression Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014361:Trypsin Inhibitors / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity*
chemical_list: D011994:Recombinant Proteins; D014361:Trypsin Inhibitors