title: Prokaryotic expression and bioactivity analysis of N-terminus domain of Pinellia ternata agglutinin using alkaline phosphatase signal peptide.
abstract: Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a two-domain monocot mannose-binding lectin. Pta-n encoding N-terminus domain of PTA (PTA-N) was fused with Escherichia coli alkaline phosphatase signal peptide (APSP) gene by polymerase chain reaction (PCR) for secretion expression. The fused nucleotide sequence apsp-pta-n was inserted into pET-28a prokaryotic expression vector by restriction enzyme digest sites (Nco I and Xho I), and then overexpressed in E. coli BL21(DE3) cells by isopropyl β-d-1-thiogalactopyranoside (IPTG) induction. Expressed APSP targeted the recombinant protein APSP-PTA-N into the periplasmic space, and then APSP was recognized and automatically cleaved by the membrane-bound signal peptidase. Ni-NTA chromatography was used for the purification and about 20 mg/L purified PTA-N was obtained. The minimum agglutination concentration of PTA-N determined by mice erythrocytes was 6.33 ± 0.47 μg/ml. The carbohydrate inhibition assay was carried out to determine the carbohydrate-binding property indicating PTA-N bound to specific sugars. The in vitro anti-proliferative activity towards human tumor cell lines and anti-fungal activity against Gibberella saubinetii were also demonstrated. Nuclear staining assay was performed to demonstrate PTA-N induced cell apoptosis. The results showed that PTA-N had significant biological functions, similar to native PTA. This strategy was the first time used to express plant mannose-binding lectin proteins and the product induced human tumor cell apoptosis, suggesting its potential application in biomedicine research.
authors.affiliation: Institute of Bioengineering, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhou
authors.forename: Yue
authors.initials: Y
authors.lastname: Huang
authors.forename: Shaowei
authors.initials: S
authors.lastname: Xu
authors.forename: Yong
authors.initials: Y
authors.lastname: Gao
authors.forename: Wenduo
authors.initials: W
authors.lastname: Chen
authors.forename: Miao
authors.initials: M
authors.lastname: Dong
authors.forename: Zhili
authors.initials: Z
authors.lastname: Yang
authors.forename: Tao
authors.initials: T
authors.lastname: Xu
mesh_terms: D000373:Agglutinins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D018506:Gene Expression Regulation, Plant; D006801:Humans; D051379:Mice; D030021:Pinellia / Q000737:chemistry / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins
chemical_list: D000373:Agglutinins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase