Affinity maturation of antibody against membrane-bound GPCR molecules
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PROVIDER: PRJEB32492 | ENA |
REPOSITORIES: ENA
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Project description:G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors, are among the most important targets against which many small molecule drugs have been developed. However, only two antibody drugs targeting GPCRs have been approved for clinical use although many antibody drugs against non-GPCR protein targets have been successfully developed for various disease indications. One of the challenges for developing anti-GPCR drugs is the high difficulty to perform affinity maturation due to their insolubility in aqueous solutions. To address this issue, CHO cell display libraries of single-chain variable fragments (scFvs) and full-length antibodies were maturated directly against vesicle probes prepared from CHO cells displaying the endothelin A receptor (ETaR) GPCR. The probe in the vesicle form ensures the physiological conformation and functional activity of the protein and avoids issues with membrane protein insolubility. The size of the vesicle had a clear effect on protein-ligand interaction; we used small-sized vesicles with low expression levels of GPCRs for the affinity maturation. Four rounds of affinity maturation combining vesicles as probes with the CHO cell display platform improved affinity by 13.58-fold for scFvs and 5.05-fold for full-length antibodies. We expect that this method will not only be used for the affinity maturation of antibodies against GPCRs but will also be used to mature antibodies for other types of proteins where the conformation/activity of which depends on the proper membrane environment.
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Project description:Analysis of the effect of G-alpha or GPCR mutation on the Arabidopsis Transcriptome Keywords: Comparitive Genome Hybridization The transcriptomes of wild type and mutants (G-alpha and GPCR) were compared in the current study. The study was carried out on four whole genome oligo arrays. The G-alpha mutant and its corresponding wild type variety were compared on a single array by means of a dual channel experiment (Cy3 and Cy5 labeling). The experiment was repeated on a different array as a flip-dye replicate and the data from both the sets analyzed further.
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