Live-cell imaging of membrane proteins by a coiled-coil labeling method—Principles and applications

Yano, Yoshiaki

doi:10.1016/j.bbamem.2019.02.009

“…The most frequently-used CC heterodimeric peptide pair is based on peptide partners that are positively (basic or K-peptide) and negatively charged (acidic or E-peptide). [9][10][11] The number of available orthogonal peptide pairs limits the range of different interactions that may be simultaneously introduced into the cell. In addition, highly positively charged peptides can interact non-specifically with biological polyanions, such as nucleic acids and charged polysaccharides.…”

Section: Introductionmentioning

confidence: 99%

A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells

Lebar

¹

,

Lainšček

²

,

Merljak

³

et al. 2020

View full text Add to dashboard Cite

Protein interactions guide the majority of cellular processes. Orthogonal hetero-specific proteinprotein interaction domains may facilitate better control of engineered biological systems. Here, we report a tunable de novo designed set of orthogonal coiled-coil (CC) peptide heterodimers (the NICP set) and its application for the regulation of diverse cellular processes, from cellular localization to transcriptional regulation. We demonstrate the application of CC pairs for multiplex localization in single cells and exploit the interaction strength and variable stoichiometry of CC peptides for tuning of gene transcription strength. A concatenated CC peptide tag (CCC-tag) was used to construct highly potent CRISPR/dCas9-based transcriptional activators and to amplify the response of light-and small-molecule inducible transcription in cell culture as well as in vivo. The NICP set and its implementations represent a valuable toolbox of minimally disruptive modules for the recruitment of versatile functional domains and regulation of cellular processes for synthetic biology.

show abstract

“…On the other hand, coiled-coil forming peptides are a pair of helical peptides that are rationally designed to show a high affinity between two or more 20 . As the proteins tethered with these peptides spontaneously form a multimer simply by mixing them in a mild environment, this technology has been used to rapidly assemble two or more proteins in vitro or in vivo 21 . Recently, an E4/K4 peptide pair was used to introduce fluorescent dye into the N-terminal region of the Q-bodies (Coiled Q-bodies); Fab and nanobody-based Q-bodies were constructed with comparable responses to conventional Q-bodies using an amber codon or Cys residue as a labeling strategy 22 .…”

Section: Introductionmentioning

confidence: 99%

Evaluation and selection of potent fluorescent immunosensors by combining fluorescent peptide and nanobodies displayed on yeast surface

Inoue

¹

,

Yasuda

²

,

Zhu

³

et al. 2021

Sci Rep

View full text Add to dashboard Cite

Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required. Here, we aimed to develop a molecular display method of nanobody-based “mini Q-bodies” by combining yeast surface display and coiled-coil forming E4/K4 peptide-based fluorescence labeling. As a result, the yeast-displayed mini Q-body recognizing the anti-cancer agent methotrexate (MTX) showed significant quenching and MTX-dependent dequenching on cells. To demonstrate the applicability of the developed method to select highly responsive mini Q-bodies, a small nanobody library consisting of 30 variants that recognize human serum albumin was used as a model. The best variant, showing a 2.4-fold signal increase, was obtained through selection by flow cytometry. Furthermore, the same nanobody prepared from Escherichia coli also worked as a mini Q-body after dye labeling. The described approach will be applied to quickly obtain well-behaved Q-bodies and other fluorescent biosensors for various targets through directed evolutionary approaches.

show abstract

“…On the other hand, coiled-coil forming peptides are a pair of helical peptides that are rationally designed to show a high a nity between two or more 20 . As the proteins tethered with these peptides spontaneously form a multimer simply by mixing them in a mild environment, this technology has been used to rapidly assemble two or more proteins in vitro or in vivo 21 . Recently, an E4/K4 peptide pair was used to introduce uorescent dye into the N-terminal region of the Qbodies (Coiled Q-bodies); Fab and nanobody-based Q-bodies were constructed with comparable responses to conventional Q-bodies using an amber codon or Cys residue as a labeling strategy 22 .…”

Section: Introductionmentioning

confidence: 99%

Evaluation and Selection of Potent Fluorescent Immunosensors by Combining Fluorescent Peptide and Nanobodies Displayed on Yeast Surface

Inoue

¹

,

Yasuda

²

,

Zhu

³

et al. 2021

Preprint

0

View full text Add to dashboard Cite

Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required. Here, we aimed to develop a molecular display method of nanobody-based “mini Q-bodies” by combining yeast surface display and coiled-coil forming E4/K4 peptide-based fluorescence labeling. As a result, the yeast-displayed mini Q-body recognizing the anti-cancer agent methotrexate (MTX) showed significant quenching and MTX-dependent dequenching on cells. To demonstrate the applicability of the developed method to select highly responsive mini Q-bodies, a small nanobody library consisting of 30 variants that recognize human serum albumin was used as a model. The best variant, showing a 2.4-fold signal increase, was obtained through selection by flow cytometry. Furthermore, the same nanobody prepared from Escherichia coli also worked as a mini Q-body after dye labeling. The described approach will be applied to quickly obtain well-behaved Q-bodies and other fluorescent biosensors for various targets through directed evolutionary approaches.

show abstract

Live-cell imaging of membrane proteins by a coiled-coil labeling method—Principles and applications

Cited by 20 publications

References 58 publications

A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells

A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells

Evaluation and selection of potent fluorescent immunosensors by combining fluorescent peptide and nanobodies displayed on yeast surface

Evaluation and Selection of Potent Fluorescent Immunosensors by Combining Fluorescent Peptide and Nanobodies Displayed on Yeast Surface

Contact Info

Product

Resources

About