关键词: 碱性磷酸酯酶；原核表达；配体印迹；同源建模, 分子对接；热点残基预测

Abstract: 【Objective】The objective of this study is to confirm the binding ability of membrane-bound alkaline phosphatase (mALP) of Plutella xyllostella with Cry1Ac toxin using prokaryotic expression and Ligand blot, and to predict toxin-receptor binding region and key amino acid binding sites (hot-spots) employed by homology modeling and docking study of Cry1Ac-mALP binding mode. It will provide a basis for the study of toxin-receptor interaction mechanism and molecular modification to enhance the activity of Cry toxin. 【Method】 The mALP of P. xyllostella full-length primers were designed and amplified by PCR. The restricted products of mALP and pET-26b (+) were ligated by T4 DNA ligase after the dual-enzyme digestion procedures. The recombinant pET-26b-mALP vectors were transferred into the Trans1-T1 phage resistant chemically competent cells, then picked clones were analyzed by PCR amplification, dual-enzyme digestion and sanger sequencing. The positive recombinant vectors (anchoring the corrected mALP gene) were transferred into the E. coli BL21 (DE3) competent cells for prokaryotic expression. The inducible expression products of mALP were transformed onto PVDF membrane. Preparation of mALP and binding activity of Cry1Ac with mALP were verified through Western blot and Ligand blot, respectively. Three-dimensional structure of mALP was predicted by homology modeling, molecular dynamics simulation and model evaluation. The toxin-receptor docking complexes were generated by using the PatchDocK and FireDock web-servers with molecular dynamics simulations. The toxin-receptor complex was analyzed to determine the interaction region and the amino acid binding sites, key amino acid residues involved in Cry toxin and ALP receptor by computer-aided alanine mutation scanning tests. 【Result】P. xyllostella mALP gene was successfully amplified, followed with the prokaryotic expression of mALP receptor protein. Binding of Cry1Ac toxin with prepared mALP protein was verified. The three-dimensional structure of mALP was successfully obtained by homology modeling, then the Cry toxin-ALP complex was determined. By the changed solvent accessible surface areas calculation and Ligplot analysis, the results showed that the domain II and domain III of Cry toxin were involved in binding to receptor, and Cry toxin and ALP were interacted mainly depending on hydrophobic and hydrogen bonding patterns. Finally, through the computer-aided alanine mutation scanning hot residues analysis, there were three key amino acid residues (376ASN, 443SER and 486SER) from Cry toxin and four key amino acid residues (452ARG, 499THR, 502TYR and 513TYR) from ALP were participated in the interaction of toxin-receptor complex, respectively. 【Conclusion】It can be determined that the mALP receptor also has the ability to bind Cry1Ac toxin by prokaryotic expression, the three-dimensional structure of mALP was predicted and the toxin-receptor binding model was studied using molecular simulation.

Key words: alkaline phosphatase (ALP), prokaryotic expression, Ligand blot, homology modeling, molecular docking, hot-spots prediction