Versatile Interacting Peptide (VIP) Tags for Labeling Proteins with Bright Chemical Reporters

Zane, Hannah K.; Doh, Julia K.; Enns, Caroline A.; Beatty, Kimberly E.

doi:10.1002/cbic.201600627

Cited by 24 publications

(43 citation statements)

References 42 publications

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“…That dimer had a reported dissociation constant (K D ) of <15 × 10 −9 M and a melting temperature (T m ) of 32°C (35). VIP Y/Z precisely labeled protein targets in living cells with various chemical reporters, including fluorophores and quantum dots (Qdots) (30).…”

Section: Resultsmentioning

confidence: 99%

“…Genetically encoded tags are widely available for FM, and a subset are compatible with SRM (1). However, most tags for FM are large (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33), which can have negative consequences on protein folding, trafficking, and function (13,14). Commonly used tags include fusions to fluorescent proteins, DNA alkyltransferases (15,16), a dehalogenase (17), or dihydrofolate reductase (18).…”

mentioning

confidence: 99%

“…We report herein a technology that enables effortless switching from FM to high-resolution EM without changing the genetically encoded tag. In 2017, we published our first versatile interacting peptide (VIP) tag, named VIP Y/Z (30). Now we present VIPER, a distinct peptide tag that has high specificity in a miniaturized size.…”

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confidence: 99%

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VIPER is a genetically encoded peptide tag for fluorescence and electron microscopy

Doh

White

Zane

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Many discoveries in cell biology rely on making specific proteins visible within their native cellular environment. There are various genetically encoded tags, such as fluorescent proteins, developed for fluorescence microscopy (FM). However, there are almost no genetically encoded tags that enable cellular proteins to be observed by both FM and electron microscopy (EM). Herein, we describe a technology for labeling proteins with diverse chemical reporters, including bright organic fluorophores for FM and electron-dense nanoparticles for EM. Our technology uses versatile interacting peptide (VIP) tags, a class of genetically encoded tag. We present VIPER, which consists of a coiled-coil heterodimer formed between the genetic tag, CoilE, and a probe-labeled peptide, CoilR. Using confocal FM, we demonstrate that VIPER can be used to highlight subcellular structures or to image receptor-mediated iron uptake. Additionally, we used VIPER to image the iron uptake machinery by correlative light and EM (CLEM). VIPER compared favorably with immunolabeling for imaging proteins by CLEM, and is an enabling technology for protein targets that cannot be immunolabeled. VIPER is a versatile peptide tag that can be used to label and track proteins with diverse chemical reporters observable by both FM and EM instrumentation.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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VIPER is a genetically encoded peptide tag for fluorescence and electron microscopy

Doh

White

Zane

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…JKD was partially funded by the Portland Chapter of Achievement Rewards for College Scientists (ARCS). The protocols described herein were originally described in two prior publications (Zane et al, 2017;Doh et al, 2018). We are grateful to our colleagues at OHSU, particularly Drs.…”

Section: Acknowledgmentsmentioning

confidence: 99%

Generation of CoilR Probe Peptides for VIPER-labeling of Cellular Proteins

Doh¹,

Tobin²,

Beatty³

2019

BIO-PROTOCOL

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Versatile Interacting Peptide (VIP) tags are a new class of genetically-encoded tag designed for imaging cellular proteins by fluorescence and electron microscopy. In 2018, we reported the VIPER tag (Doh et al., 2018), which contains two elements: a genetically-encoded peptide tag (i.e., CoilE) and a probe peptide (i.e., CoilR). These two peptides deliver contrast to a protein of interest by forming a specific, high-affinity heterodimer. The probe peptide was designed with a single cysteine residue for site-specific modification via thiol-maleimide chemistry. This feature can be used to attach a variety of biophysical reporters to the peptide, including bright fluorophores for fluorescence microscopy or electron-dense nanoparticles for electron microscopy. In this Bio-Protocol, we describe our methods for expressing and purifying recombinant CoilR. Additionally, we describe protocols for making fluorescent or biotinylated probe peptides for labeling CoilE-tagged cellular proteins. This protocol is complemented by two other Bio-Protocols outlining the use of VIPER (Doh et al., 2019a and 2019b).

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“…The VIP probe peptides were designed to have a single cysteine residue that acts as a bioconjugation site for attachment to fluorophores or EM labels. Our first VIP tag, VIP Y/Z (comprised of CoilY and CoilZ), enabled selective fluorophore labeling of proteins on living cells [6].…”

mentioning

confidence: 99%