Expression and purification of a novel mannose-binding lectin from Pinellia ternata.
Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes to a dilution similar to that determined for "native" lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and purification of biologically active recombinant PTA from E. coli.
Lin
Juan
J
State Key Laboratory of Genetic Engineering, School of Life Sciences, Morgan-Tan International Center for Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Fudan University, Shanghai 200433, People's Republic of China.
Yao
Jianhong
J
Zhou
Xuanwei
X
Sun
Xiaofen
X
Tang
Kexuan
K
D000818:Animals; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006384:Hemagglutination / Q000187:drug effects; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D037241:Mannose-Binding Lectins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D030021:Pinellia / Q000737:chemistry*; D037121:Plant Lectins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology
D037241:Mannose-Binding Lectins; C047938:Pinellia ternata lectin; D037121:Plant Lectins; D011994:Recombinant Proteins