Protein Expression and Purification, Vol.89, No.1, 84-91, 2013
Prokaryotic expression and bioactivity analysis of N-terminus domain of Pinellia ternata agglutinin using alkaline phosphatase signal peptide
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 coil 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 beta-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 mu g/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. (C) 2013 Elsevier Inc. All rights reserved.