Expanding the Chemogenetic Toolbox by Circular Permutation.

abstract

• To expand the repertoire of chemogenetic tools tailored for molecular and cellular engineering, we describe herein the design of cpRAPID as a circularly permuted rapamycin-inducible dimerization system composed of the canonical FK506-binding protein (FKBP) and circular permutants of FKBP12-rapamycin binding domain (cpFRB). By permuting the topology of the four helices within FRB, we have created cpFRB-FKBP pairs that respond to ligand with varying activation kinetics and dynamics. The cpRAPID system enables chemical-controllable subcellular redistribution of proteins, as well as inducible transcriptional activation when coupled with the CRISPR activation (CRISPRa) technology to induce a GFP reporter and endogenous gene expression. We have further demonstrated the use of cpRAPID to generate chemically switchable split nanobody (designated Chessbody) for ligand-gated antigen recognition in living cells. Collectively, the circular permutation approach offers a powerful means for diversifying the chemogenetics toolbox to benefit the burgeoning synthetic biology field.

authors

• Zhou, Yubin

published proceedings

• J Mol Biol

altmetric score

• 1.25

author list (cited authors)

• Lee, Y., He, L., & Zhou, Y.

citation count

• 2

complete list of authors

• Lee, Yi-Tsang||He, Lian||Zhou, Yubin

publication date

• January 2020

publisher

• Elsevier  Publisher

published in

• Journal of Molecular Biology  Journal

keywords

• Antibody Engineering
• CRISPR
• Chemogenetics
• Cloning, Molecular
• HEK293 Cells
• Hela Cells
• Humans
• Ligands
• Nanobody
• Protein Domains
• Protein Engineering
• Protein Structure, Secondary
• Recombinant Proteins
• Sirolimus
• Synthetic Biology
• Tacrolimus Binding Protein 1A
• Tacrolimus Binding Proteins

Digital Object Identifier (DOI)

• 10.1016/j.jmb.2020.03.033

start page

• 3127

end page

• 3136

volume

• 432

issue

• 10

URL

• http://dx.doi.org/10.1016/j.jmb.2020.03.033