The knowledge of interactions among functional proteins helps researchers understand disease mechanisms and design potential strategies for treatment.As a general approach,the fluorescent and affinity tags were employ...The knowledge of interactions among functional proteins helps researchers understand disease mechanisms and design potential strategies for treatment.As a general approach,the fluorescent and affinity tags were employed for exploring this field by labeling the Protein of Interest(POI).However,the autofluorescence and weak binding strength significantly reduce the accuracy and specificity of these tags.Conversely,HaloTag,a novel self-labeling enzyme(SLE)tag,could quickly form a covalent bond with its ligand,enabling fast and specific labeling of POI.These desirable features greatly increase the accuracy and specificity,making the HaloTag a valuable system for various applications ranging from imaging to immobilization of POI.Notably,the HaloTag technique has already been successfully employed in a series of studies with excellent efficiency.In this review,we summarize the development of HaloTag and recent advanced investigations associated with HaloTag,including in vitro imaging(e.g.,POI imaging,cellular condition monitoring,microorganism imaging,system development),in vivo imaging,biomolecule immobilization(e.g.,POI collection,protein/nuclear acid interaction and protein structure analysis),targeted degradation(e.g.,L-AdPROM),and more.We also present a systematic discussion regarding the future direction and challenges of the HaloTag technique.展开更多
Red fluorescent proteins with large Stokes shift(LSS-RFPs)are advantageous for multicolor imaging applications that allow simultaneous visualizations of multiple biological events.But it is difficult to develop LSS-RF...Red fluorescent proteins with large Stokes shift(LSS-RFPs)are advantageous for multicolor imaging applications that allow simultaneous visualizations of multiple biological events.But it is difficult to develop LSS-RFPs by extending the emission wavelength of RFPs to far-red region.Here,we employed Forster resonance energy transfer(FRET)strategy to engineer the far-red fluorescent proteins with large Stokes shift.LSS-m Apple and LSS-mCherry were constructed by fusing HaloTag to m Apple and mCherry,allowing the fluorophore TMSi R to be connected to these RFPs.FRET between RFPs and TMSi R enabled them to apply the excitation of donor RFPs to emit far-red fluorescence of acceptor TMSi R.The Stokes shifts of LSS-m Apple and LSS-mCherry were 97 nm and 75 nm,respectively.The high FRET efficiency of LSS-mCherry(E_(FRET)=83.7%)can greatly reduce the fluorescence from the donor channel,which did not affect co-imaging with mCherry.In addition,LSS-mCherry also showed excellent photostability(t_(1/2)=449.3 s),enabling stable confocal fluorescence imaging for 15 min under continuous strong excitation.Furthermore,LSS-mCherry was applied for fluorescence labeling and imaging of the nucleus,mitochondria,lysosomes,and endoplasmic reticulum in living cells.Finally,we applied LSS-mCherry to perform multi-color bioimaging of 2–4 channels,and there was no obvious crosstalk between these channels.展开更多
GPCRs are dominant targets for approved drugs and the discovery of lead compound targeting them is still challengeable.Affinity-based screening technique is a promising platform to uncover GPCR ligands.However,the int...GPCRs are dominant targets for approved drugs and the discovery of lead compound targeting them is still challengeable.Affinity-based screening technique is a promising platform to uncover GPCR ligands.However,the intrinsic activities of them are seldom simultaneously determined during the screening.Taking beta2-adrenoceptor(β2AR)as a probe,this work created a strategy for screening GPCR ligands with simultaneous characterization of their downstream G protein binding responses associated with GTP.The strategy included(i)the design and expression of a protein miniature formed byβ2AR and G proteinα-subunit(Gαs)using circularly permuted HaloTag(cpHalo)as a flexible linker;(ii)immobilization of the miniature onto silica gel by a click dehalogenation reaction;(iii)systematic characterization of the immobilized miniature by fluorescent and chromatographic studies,and(iv)simulating of ligand-inducedβ2AR-Gαs signaling cascade by chromatographic assays using GTP as an indicator.The immobilized miniature exhibited specificity toβ2AR and Gαs antibodies and ligands.The specificity is stable at least within fifteen days with the variation less than 1%.The intrinsic activities ofβ2AR ligands were distinguished by the changes of GTP chromatographic behaviors on Gαs-cpHalo-β2AR column.Agonists strengthened the binding affinity and kinetics of GTP with Gαs,while antagonist did not give any effect on them.With the intrinsic activity evaluation,we believe,it will improve the attributes of chromatographic methods for drug discovery efforts with minimizing false-positive results.展开更多
Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins （RBPs） by the UV-crosslinking and immunoprecipitation （CLIP） represents one o...Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins （RBPs） by the UV-crosslinking and immunoprecipitation （CLIP） represents one of the most powerful methods to map protein RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method （GoldCLIP） that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein （PTB）, a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.展开更多
基金The authors are grateful for financial support from the University of Wisconsin—Madison and National Institutes of Health(P30CA014520).
文摘The knowledge of interactions among functional proteins helps researchers understand disease mechanisms and design potential strategies for treatment.As a general approach,the fluorescent and affinity tags were employed for exploring this field by labeling the Protein of Interest(POI).However,the autofluorescence and weak binding strength significantly reduce the accuracy and specificity of these tags.Conversely,HaloTag,a novel self-labeling enzyme(SLE)tag,could quickly form a covalent bond with its ligand,enabling fast and specific labeling of POI.These desirable features greatly increase the accuracy and specificity,making the HaloTag a valuable system for various applications ranging from imaging to immobilization of POI.Notably,the HaloTag technique has already been successfully employed in a series of studies with excellent efficiency.In this review,we summarize the development of HaloTag and recent advanced investigations associated with HaloTag,including in vitro imaging(e.g.,POI imaging,cellular condition monitoring,microorganism imaging,system development),in vivo imaging,biomolecule immobilization(e.g.,POI collection,protein/nuclear acid interaction and protein structure analysis),targeted degradation(e.g.,L-AdPROM),and more.We also present a systematic discussion regarding the future direction and challenges of the HaloTag technique.
基金supported by the National Natural Science Foundation of China(Nos.22225806,22078314,22278394,22378385)Dalian Institute of Chemical Physics(Nos.DICPI202142,DICPI202436)。
文摘Red fluorescent proteins with large Stokes shift(LSS-RFPs)are advantageous for multicolor imaging applications that allow simultaneous visualizations of multiple biological events.But it is difficult to develop LSS-RFPs by extending the emission wavelength of RFPs to far-red region.Here,we employed Forster resonance energy transfer(FRET)strategy to engineer the far-red fluorescent proteins with large Stokes shift.LSS-m Apple and LSS-mCherry were constructed by fusing HaloTag to m Apple and mCherry,allowing the fluorophore TMSi R to be connected to these RFPs.FRET between RFPs and TMSi R enabled them to apply the excitation of donor RFPs to emit far-red fluorescence of acceptor TMSi R.The Stokes shifts of LSS-m Apple and LSS-mCherry were 97 nm and 75 nm,respectively.The high FRET efficiency of LSS-mCherry(E_(FRET)=83.7%)can greatly reduce the fluorescence from the donor channel,which did not affect co-imaging with mCherry.In addition,LSS-mCherry also showed excellent photostability(t_(1/2)=449.3 s),enabling stable confocal fluorescence imaging for 15 min under continuous strong excitation.Furthermore,LSS-mCherry was applied for fluorescence labeling and imaging of the nucleus,mitochondria,lysosomes,and endoplasmic reticulum in living cells.Finally,we applied LSS-mCherry to perform multi-color bioimaging of 2–4 channels,and there was no obvious crosstalk between these channels.
基金National Natural Science Foundation of China(Nos.22374116,22074118,82174088)Natural Science Basic Research Program of Shaanxi(2024JC-TBZC-21)Shaanxi Administration of Traditional Chinese Medicine(No.2022-SLRH-YQ-007)。
文摘GPCRs are dominant targets for approved drugs and the discovery of lead compound targeting them is still challengeable.Affinity-based screening technique is a promising platform to uncover GPCR ligands.However,the intrinsic activities of them are seldom simultaneously determined during the screening.Taking beta2-adrenoceptor(β2AR)as a probe,this work created a strategy for screening GPCR ligands with simultaneous characterization of their downstream G protein binding responses associated with GTP.The strategy included(i)the design and expression of a protein miniature formed byβ2AR and G proteinα-subunit(Gαs)using circularly permuted HaloTag(cpHalo)as a flexible linker;(ii)immobilization of the miniature onto silica gel by a click dehalogenation reaction;(iii)systematic characterization of the immobilized miniature by fluorescent and chromatographic studies,and(iv)simulating of ligand-inducedβ2AR-Gαs signaling cascade by chromatographic assays using GTP as an indicator.The immobilized miniature exhibited specificity toβ2AR and Gαs antibodies and ligands.The specificity is stable at least within fifteen days with the variation less than 1%.The intrinsic activities ofβ2AR ligands were distinguished by the changes of GTP chromatographic behaviors on Gαs-cpHalo-β2AR column.Agonists strengthened the binding affinity and kinetics of GTP with Gαs,while antagonist did not give any effect on them.With the intrinsic activity evaluation,we believe,it will improve the attributes of chromatographic methods for drug discovery efforts with minimizing false-positive results.
基金supported in part by grants from the Ministry of Science and Technology of China(Grant No.2017YFA0504200 to YY,Grant Nos.2012CB910502 and 2011CB966304 to JM)the National Natural Science Foundation of China(Grant Nos.91640105 and 31770875 to YY,Grant No.31230041 to JM,and Grant Nos.91640115 and 31670827 to YZ)
文摘Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins （RBPs） by the UV-crosslinking and immunoprecipitation （CLIP） represents one of the most powerful methods to map protein RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method （GoldCLIP） that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein （PTB）, a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.
