Synthetic Polymer Affinity Ligand for Bacillus thuringiensis (Bt) Cry1Ab/Ac Protein: The Use of Biomimicry Based on the Bt Protein–Insect Receptor Binding Mechanism
2018
Liu, Mingming | Huang, Rong | Weisman, Adam | Yu, Xiaoyang | Lee, Shih-Hui | Chen, Yalu | Huang, Chao | Hu, Senhua | Chen, Xiuhua | Tan, Wenfeng | Liu, Fan | Chen, Hao | Shea, Kenneth J.
We report a novel strategy for creating abiotic Bacillus thuringiensis (Bt) protein affinity ligands by biomimicry of the recognition process that takes place between Bt Cry1Ab/Ac proteins and insect receptor cadherin-like Bt-R₁ proteins. Guided by this strategy, a library of synthetic polymer nanoparticles (NPs) was prepared and screened for binding to three epitopes ²⁸⁰FRGSAQGIEGS²⁹⁰, ³⁶⁸RRPFNIGINNQQ³⁷⁹ and ⁴³⁶FRSGFSNSSVSIIR⁴⁴⁹ located in loop α8, loop 2 and loop 3 of domain II of Bt Cry1Ab/Ac proteins. A negatively charged and hydrophilic nanoparticle (NP12) was found to have high affinity to one of the epitopes, ³⁶⁸RRPFNIGINNQQ³⁷⁹. This same NP also had specific binding ability to both Bt Cry1Ab and Bt Cry1Ac, proteins that share the same epitope, but very low affinity to Bt Cry2A, Bt Cry1C and Bt Cry1F closely related proteins that lack epitope homology. To locate possible NP-Bt Cry1Ab/Ac interaction sites, NP12 was used as a competitive inhibitor to block the binding of ⁸⁶⁵NITIHITDTNNK⁸⁷⁶, a specific recognition site in insect receptor Bt-R₁, to ³⁶⁸RRPFNIGINNQQ³⁷⁹. The inhibition by NP12 reached as high as 84%, indicating that NP12 binds to Bt Cry1Ab/Ac proteins mainly via ³⁶⁸RRPFNIGINNQQ³⁷⁹. This epitope region was then utilized as a “target” or “bait” for the separation and concentration of Bt Cry1Ac protein from the extract of transgenic Bt cotton leaves by NP12. This strategy, based on the antigen-receptor recognition mechanism, can be extended to other biotoxins and pathogen proteins when designing biomimic alternatives to natural protein affinity ligands.
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