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New Progress Achieved in Creating Abiotic Bt Protein Affinity Ligands

An important headway was made in creating abiotic Bacillus thuringiensis (Bt) protein affinity ligands by the research group of soil minerals and environment from the College of Resources and Environmental Sciences of HZAU. The achievements were published in the Journal of American Chemical Society, the title of which was Synthetic Polymer Affinity Ligand for Bacillus thuringiensis (Bt) Cry1Ab/Ac Protein: The Use of Biomimicry Based on the Bt Protein-Insect Receptor Binding Mechanism. Pro. Liu Mingming of the College of Resources and Environmental Sciences is the first author and corresponding author of the paper.
The research group report a novel strategy for creating abiotic 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, which is the next research direction of the group.

This project was sponsored by the National Natural Science Foundation of China (No.21577044 & No.21177047), the Ministry of Education’s New Century Talents Program (Grant No. NCET-13-0808), the State Key Project for the Cultivation of New Varieties of Genetically Modified Organisms (2016ZX08001001), and the HZAU’s Independent Innovation Technology Fund (No.2014PY019 & No.2013PY138).

Translated by Ding Xubin