An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification

被引:21
作者
Cai, Hongmin [1 ]
Yao, Hebang [1 ]
Li, Tingting [1 ]
Hutter, Cedric A. J. [2 ]
Li, Yanfang [1 ]
Tang, Yannan [1 ]
Seeger, Markus A. [2 ]
Li, Dianfan [1 ]
机构
[1] Chinese Acad Sci, Univ Chinese Acad Sci, Shanghai Inst Biochem & Cell Biol, CAS Ctr Excellence Mol Cell Sci,Natl Ctr Prot Sci, 320 Yueyang Rd, Shanghai 200031, Peoples R China
[2] Univ Zurich, Inst Med Microbiol, Zurich, Switzerland
基金
中国国家自然科学基金;
关键词
SIZE-EXCLUSION CHROMATOGRAPHY; HIGH-THROUGHPUT EXPRESSION; COUPLED RECEPTORS; CRYSTAL-STRUCTURE; ESCHERICHIA-COLI; SCREENING METHOD; CRYSTALLIZATION; OVEREXPRESSION; THERMOSTABILIZATION; CHALLENGES;
D O I
10.1038/s42003-020-01478-z
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. An ideal reporter should be stable itself and provide high sensitivity and yield. Here, we demonstrate that a coral (Galaxea fascicularis) thermostable GFP (TGP) is by such reasons an improved tag compared to the conventional jellyfish GFPs. TGP faithfully reports membrane protein stability at temperatures near 90 degrees C (20-min heating). By contrast, the limit for the two popular GFPs is 64 degrees C and 74 degrees C. Replacing GFPs with TGP increases yield for all four test membrane proteins in four expression systems. To establish TGP as an affinity tag for membrane protein purification, several high-affinity synthetic nanobodies (sybodies), including a non-competing pair, are generated, and the crystal structure of one complex is solved. Given these advantages, we anticipate that TGP becomes a widely used tool for membrane protein structural studies. In this work, the authors demonstrate that a coral thermostable GFP (TGP) is an improved tag compared to conventional GFPs. In addition to reporting melting point stability at temperatures near 90 degrees C, its fusion also helps increase expression levels of test membrane proteins. They further generated synthetic nanobodies against TGP to facilitate purification.
引用
收藏
页数:16
相关论文
共 91 条
  • [61] Phaser crystallographic software
    McCoy, Airlie J.
    Grosse-Kunstleve, Ralf W.
    Adams, Paul D.
    Winn, Martyn D.
    Storoni, Laurent C.
    Read, Randy J.
    [J]. JOURNAL OF APPLIED CRYSTALLOGRAPHY, 2007, 40 : 658 - 674
  • [62] Rapid determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins
    Moreau, Morgane J. J.
    Morin, Isabelle
    Askin, Samuel. P.
    Cooper, Alanna
    Moreland, Nicole J.
    Vasudevan, Subhash G.
    Schaeffer, Patrick M.
    [J]. RSC ADVANCES, 2012, 2 (31): : 11892 - 11900
  • [63] A general protocol for the crystallization of membrane proteins for X-ray structural investigation
    Newby, Zachary E. R.
    O'Connell, Joseph D., III
    Gruswitz, Franz
    Hays, Franklin A.
    Harries, William E. C.
    Harwood, Ian M.
    Ho, Joseph D.
    Lee, John K.
    Savage, David F.
    Miercke, Larry J. W.
    Stroud, Robert M.
    [J]. NATURE PROTOCOLS, 2009, 4 (05) : 619 - 637
  • [64] An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins
    Nji, Emmanuel
    Chatzikyriakidou, Yurie
    Landreh, Michael
    Drew, David
    [J]. NATURE COMMUNICATIONS, 2018, 9
  • [65] Structure of a CLC chloride ion channel by cryo-electron microscopy
    Park, Eunyong
    Ampbell, Ernest B. C.
    MacKinnon, Roderick
    [J]. NATURE, 2017, 541 (7638) : 500 - 505
  • [66] Membrane Protein Crystallisation: Current Trends and Future Perspectives
    Parker, Joanne L.
    Newstead, Simon
    [J]. NEXT GENERATION IN MEMBRANE PROTEIN STRUCTURE DETERMINATION, 2016, 922 : 61 - 72
  • [67] Method to increase the yield of eukaryotic membrane protein expression in Saccharomyces cerevisiae for structural and functional studies
    Parker, Joanne L.
    Newstead, Simon
    [J]. PROTEIN SCIENCE, 2014, 23 (09) : 1309 - 1314
  • [68] Improving the stability and function of purified ABCB1 and ABCA4: The influence of membrane lipids
    Pollock, Naomi L.
    McDevitt, Christopher A.
    Collins, Richard
    Niesten, Petronella H. M.
    Prince, Stephen
    Kerr, Ian D.
    Ford, Robert C.
    Callaghan, Richard
    [J]. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 2014, 1838 (01): : 134 - 147
  • [69] An Improved Strategy for Fluorescent Tagging of Membrane Proteins for Overexpression and Purification in Mammalian Cells
    Rana, Mitra S.
    Wang, Xiyu
    Banerjee, Anirban
    [J]. BIOCHEMISTRY, 2018, 57 (49) : 6741 - 6751
  • [70] Salt Effects on the Conformational Stability of the Visual G-Protein-Coupled Receptor Rhodopsin
    Reyes-Alcaraz, Arfaxad
    Martinez-Archundia, Marlet
    Ramon, Eva
    Garriga, Pere
    [J]. BIOPHYSICAL JOURNAL, 2011, 101 (11) : 2798 - 2806