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Crystal structure of circular permuted RoCBM21 (CP90): dimerisation and proximity of binding sites.

ABSTRACT: Glucoamylases, containing starch-binding domains (SBD), have a wide range of scientific and industrial applications. Random mutagenesis and DNA shuffling of the gene encoding a starch-binding domain have resulted in only minor improvements in the affinities of the corresponding protein to their ligands, whereas circular permutation of the RoCBM21 substantially improved its binding affinity and selectivity towards longer-chain carbohydrates. For the study reported herein, we used a standard soluble ligand (amylose EX-I) to characterize the functional and structural aspects of circularly permuted RoCBM21 (CP90). Site-directed mutagenesis and the analysis of crystal structure reveal the dimerisation and an altered binding path, which may be responsible for improved affinity and altered selectivity of this newly created starch-binding domain. The functional and structural characterization of CP90 suggests that it has significant potential in industrial applications.

SUBMITTER: Stephen P

PROVIDER: S-EPMC3511584 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Crystal structure of circular permuted RoCBM21 (CP90): dimerisation and proximity of binding sites.

Stephen Preyesh P Cheng Kuo-Chang KC Lyu Ping-Chiang PC

PloS one 20121130 11

Glucoamylases, containing starch-binding domains (SBD), have a wide range of scientific and industrial applications. Random mutagenesis and DNA shuffling of the gene encoding a starch-binding domain have resulted in only minor improvements in the affinities of the corresponding protein to their ligands, whereas circular permutation of the RoCBM21 substantially improved its binding affinity and selectivity towards longer-chain carbohydrates. For the study reported herein, we used a standard solub ...[more]

PMID: 23226294

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Project description:Rationale: Circular RNA (circRNA) therapeutics hold great promise as an iteration strategy in messenger RNA (mRNA) therapeutics due to their inherent stability and durable protein translation capability. Nevertheless, the efficiency of RNA circularization remains a significant constraint, particularly in establishing large-scale manufacturing processes for producing highly purified circRNAs. Hence, it is imperative to develop a universal and more efficient RNA circularization system when considering synthetic circRNAs as therapeutic agents with prospective clinical applications. Methods: We initially developed a chimeric RNA circularization system based on the original permuted intron-exon (PIE) and subsequently established a high-performance liquid chromatography (HPLC) method to obtain highly purified circRNAs. We then evaluated their translational ability and immunogenicity. The circRNAs expressing human papillomavirus (HPV) E7 peptide (43-62aa) and dimerized receptor binding domain (dRBD) from SARS-CoV-2 were encapsulated within lipid nanoparticles (LNPs) as vaccines, followed by an assessment of the in vivo efficacy through determination of antigen-specific T and B cell responses, respectively. Results: We have successfully developed a universal chimeric permuted intron-exon system (CPIE) through engineering of group I self-splicing introns derived from Anabaena pre-tRNALeu or T4 phage thymidylate (Td) synthase gene. Within CPIE, we have effectively enhanced RNA circularization efficiency. By utilizing size exclusion chromatography, circRNAs were effectively separated, which exhibit low immunogenicity and sustained potent protein expression property. In vivo data demonstrate that the constructed circRNA vaccines can elicit robust immune activation (B cell and/or T cell responses) against tumor or SARS-CoV-2 and its variants in mouse models. Conclusions: Overall, we provide an efficient and universal system to synthesize circRNA in vitro, which has extensive application prospect for circRNA therapeutics.

Dataset Information

Crystal structure of circular permuted RoCBM21 (CP90): dimerisation and proximity of binding sites.

Publications

Crystal structure of circular permuted RoCBM21 (CP90): dimerisation and proximity of binding sites.

Similar Datasets

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