Advertisement

Subcellular Localization of Intracellular Human Proteins by Construction of Tagged Fusion Proteins and Transient Expression in COS-7 Cells

  • John E. Collins
Part of the Methods in Molecular Biology™ book series (MIMB, volume 439)

Abstract

Identifying the subcellular compartment of a protein is an important step toward assigning protein function. Starting with a clone containing the open reading frame (ORF) of interest, it is possible to attach a variety of short amino acid tags or fluorescent proteins and detect the location of the protein, after transfection into a cell line, using fluorescent microscopy. By collecting data from various expression clone constructs, using a range of cell lines and double labeling with cellular compartment markers, a picture of the localization of a gene can be built up. This chapter describes how to obtain the ORF clone for your gene of interest, clone it into your choice of mammalian expression vector or vectors, transiently transfect for visualization, and where to get started when interpreting the results.

Keywords

subcellular localization open reading frame mammalian expression clone fluorescent microscopy 

Notes

Acknowledgments

We thank Begoña Aguado, Carol Edwards, Catherine Taylor, Charmain Wright, Frida Andersson, Gözde Akdeniz, James Grinham, Matthew Davis, and Meera Mallya for assistance with developing the protocols. We also thank Catherine Taylor and Ian Dunham for critically reading the manuscript. This work was supported by the Wellcome Trust.

References

  1. 1.
    1. Vidal M (2001) A biological atlas of functional maps. Cell 104:333–339CrossRefPubMedGoogle Scholar
  2. 2.
    2. Simpson JC, Pepperkok R (2006) The subcellular localization of the mammalian proteome comes a fraction closer. Genome Biol 7:222CrossRefPubMedGoogle Scholar
  3. 3.
    3. Southern JA, Young DF, Heaney F, Baumgartner WK, Randall RE (1991) Identification of an epitope on the P and V proteins of simian virus 5 that distinguishes between two isolates with different biological characteristics. J Gen Virol. 72, Part 7:1551–1557CrossRefPubMedGoogle Scholar
  4. 4.
    4. Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10:1788–1795CrossRefPubMedGoogle Scholar
  5. 5.
    5. Walhout AJ, Temple GF, Brasch MA, Hartley, JL, Lorson MA, van den Heuvel S, Vidal M (2000) GATEWAY recombinational cloning: Application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol 328:575–592CrossRefPubMedGoogle Scholar
  6. 6.
    6. Mehrle A, Rosenfelder H, Schupp I, del Val C, Arlt D, Hahne F, Bechtel S, Simpson J, Hofmann O, Hide W et al (2006) The LIFEdb database in 2006. Nucleic Acids Res 34: D415–418CrossRefPubMedGoogle Scholar
  7. 7.
    7. Simpson JC, Wellenreuther R, Poustka A, Pepperkok R, Wiemann S (2000) Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing. EMBO Rep 1:287–292CrossRefPubMedGoogle Scholar
  8. 8.
    8. Fink JL, Aturaliya RN, Davis MJ, Zhang F, Hanson K, Teasdale MS, Kai C, Kawai J, Carninci P, Hayashizaki Y et al (2006) LOCATE: A mouse protein subcellular localization database. Nucleic Acids Res 34:D213–D217CrossRefPubMedGoogle Scholar
  9. 9.
    9. Temple G, Lamesch P, Milstein S, Hill DE, Wagner L, Moore T, Vidal M (2006) From genome to proteome: Developing expression clone resources for the human genome. Hum Mol Genet 15, Spec No 1:R31–R43CrossRefPubMedGoogle Scholar
  10. 10.
    10. Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K et al (2004) Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat Genet 36:40–45CrossRefPubMedGoogle Scholar
  11. 11.
    11. Strausberg, RL, Feingold EA, Grouse LH, Derge JG, Klausner RD, Collins FS, Wagner L, Shenmen CM, Schuler GD, Altschul SF et al (2002) Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proc Natl Acad Sci USA 99:16899–16903CrossRefPubMedGoogle Scholar
  12. 12.
    12. Wiemann S, Arlt D, Huber W, Wellenreuther R, Schleeger S, Mehrle A, Bechtel S, Sauermann M, Korf U, Pepperkok R et al (2004) From ORFeome to biology: A functional genomics pipeline. Genome Res 14:2136–2144CrossRefPubMedGoogle Scholar
  13. 13.
    13. Nakajima D, Saito K, Yamakawa H, Kikuno RF, Nakayama M, Ohara R, Okazaki N, Koga H, Nagase T, Ohara O (2005) Preparation of a set of expression-ready clones of mammalian long cDNAs encoding large proteins by the ORF trap cloning method. DNA Res 12:257–267CrossRefPubMedGoogle Scholar
  14. 14.
    14. Rual JF, Hirozane-Kishikawa T, Hao T, Bertin N, Li S, Dricot A, Li N, Rosenberg J, Lamesch P, Vidalain PO et al (2004) Human ORFeome version 1.1: A platform for reverse proteomics. Genome Res 14:2128–2135CrossRefPubMedGoogle Scholar
  15. 15.
    15. Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P et al (2004) The status, quality, and expansion of the NIH full-length cDNA project: The Mammalian Gene Collection (MGC). Genome Res 14:2121–2127CrossRefPubMedGoogle Scholar
  16. 16.
    16. Collins JE, Wright CL, Edwards CA, Davis MP, Grinham JA, Cole CG, Goward ME, Aguado B, Mallya M, Mokrab Y et al (2004) A genome annotation-driven approach to cloning the human ORFeome. Genome Biol 5:R84CrossRefPubMedGoogle Scholar
  17. 17.
    17. Takagi M, Nishiok, M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M, Imanaka T (1997) Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Appl Environ. Microbiol 63:4504–4510PubMedGoogle Scholar
  18. 18.
    18. Kozak M (1999) Initiation of translation in prokaryotes and eukaryotes. Gene 234:187–208CrossRefPubMedGoogle Scholar
  19. 19.
    19. Bendtsen JD, Nielsen H, von Heijne G, Brunak,S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795CrossRefPubMedGoogle Scholar
  20. 20.
    20. Nakai K, Horton P (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24:34–36CrossRefPubMedGoogle Scholar
  21. 21.
    21. Glory E, Murphy R. (2007) Automated subcellular location determination and high-throughput microscopy. Dev Cell 12:7–16CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • John E. Collins
    • 1
  1. 1.Wellcome Trust Sanger InstituteCambridgeUK

Personalised recommendations