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Galectins pp 37-49 | Cite as

Cloning, Expression, and Purification of Galectins for In Vitro Studies

  • Paul A. Poland
  • Carol L. Kinlough
  • Rebecca P. HugheyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1207)

Abstract

Galectins are best known for their ability to bind glycoconjugates containing β-galactose, but classification of these small proteins within the galectin family is also defined by amino acid homology within structural domains and exon/intron junctions within genes. As galectins are expressed by organisms as diverse as some fungi, C. elegans, fish, birds, and mammals, and biological activities attributed to galectins are equally diverse, it becomes essential to identify, clone, and characterize galectins from many sources. Glutathione S-transferase (GST) fused to the amino-terminus of galectin cDNAs has proven to be especially useful for preparation of recombinant galectins in bacteria for use on glycan arrays, in experiments with cultured or isolated cells, and in pull-down assays with immunopurified glycoproteins. Many galectins are stabilized by reducing reagents, such that binding and elution of GST-galectins from glutathione-conjugated Sepharose with excess glutathione is both efficient and innocuous. The ability to bind and elute GST-galectins from lactose-conjugated Sepharose with excess lactose provides a relatively easy means to insure that galectins are competent for glycoconjugate binding prior to experimentation. This chapter focuses primarily on the varied approaches to use GST-galectin binding to glutathione- and lactose-conjugated Sepharose to purify recombinant galectins and then develop effective experimental protocols to characterize the specificity, interactions, and function of galectins cloned from any source. We provide one example where a pull-down assay with all the GST-tagged canine galectins reveals that the C-terminal carbohydrate recognition domain of galectin-9 (Gal-9C) specifically recognizes the glycan-dependent apical targeting signal from the glycoprotein MUC1.

Key words

Galectin GST-galectin Recombinant galectin Pull-down assay 

Notes

Acknowledgments

This work was funded by grants to RPH by National Institutes of Health (DK054787) and Genzyme Renal Innovations Program.

References

  1. 1.
    Potter BA, Hughey RP, Weisz OA (2006) Role of N- and O-glycans in polarized biosynthetic sorting. Am J Physiol Cell Physiol 290(1):C1–C10. doi: 10.1152/ajpcell.00333.2005 PubMedCrossRefGoogle Scholar
  2. 2.
    Mattila PE, Kinlough CL, Bruns JR, Weisz OA, Hughey RP (2009) MUC1 traverses apical recycling endosomes along the biosynthetic pathway in polarized MDCK cells. Biol Chem 390(7):551–556. doi: 10.1515/BC.2009.088 PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Delacour D, Gouyer V, Zanetta JP, Drobecq H, Leteurtre E, Grard G, Moreau-Hannedouche O, Maes E, Pons A, Andre S, Le Bivic A, Gabius HJ, Manninen A, Simons K, Huet G (2005) Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells. J Cell Biol 169(3):491–501. doi: 10.1083/jcb.200407073 PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Stechly L, Morelle W, Dessein AF, Andre S, Grard G, Trinel D, Dejonghe MJ, Leteurtre E, Drobecq H, Trugnan G, Gabius HJ, Huet G (2009) Galectin-4-regulated delivery of glycoproteins to the brush border membrane of enterocyte-like cells. Traffic 10(4):438–450. doi: 10.1111/j.1600-0854.2009.00882.x PubMedCrossRefGoogle Scholar
  5. 5.
    Delacour D, Cramm-Behrens CI, Drobecq H, Le Bivic A, Naim HY, Jacob R (2006) Requirement for galectin-3 in apical protein sorting. Curr Biol 16(4):408–414. doi: 10.1016/j.cub.2005.12.046 PubMedCrossRefGoogle Scholar
  6. 6.
    Delacour D, Greb C, Koch A, Salomonsson E, Leffler H, Le Bivic A, Jacob R (2007) Apical sorting by galectin-3-dependent glycoprotein clustering. Traffic 8(4):379–388. doi: 10.1111/j.1600-0854.2007.00539.x PubMedCrossRefGoogle Scholar
  7. 7.
    Delacour D, Koch A, Ackermann W, Eude-Le Parco I, Elsasser HP, Poirier F, Jacob R (2008) Loss of galectin-3 impairs membrane polarisation of mouse enterocytes in vivo. J Cell Sci 121(Pt 4):458–465. doi: 10.1242/jcs.020800 PubMedCrossRefGoogle Scholar
  8. 8.
    Kinlough CL, Poland PA, Gendler SJ, Mattila PE, Mo D, Weisz OA, Hughey RP (2011) Core-glycosylated mucin-like repeats from MUC1 are an apical targeting signal. J Biol Chem 286(45):39072–39081. doi: 10.1074/jbc.M111.289504 PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Poland PA, Rondanino C, Kinlough CL, Heimburg-Molinaro J, Arthur CM, Stowell SR, Smith DF, Hughey RP (2011) Identification and characterization of endogenous galectins expressed in Madin Darby canine kidney cells. J Biol Chem 286(8):6780–6790. doi: 10.1074/jbc.M110.179002 PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Friedrichs J, Torkko JM, Helenius J, Teravainen TP, Fullekrug J, Muller DJ, Simons K, Manninen A (2007) Contributions of galectin-3 and -9 to epithelial cell adhesion analyzed by single cell force spectroscopy. J Biol Chem 282(40):29375–29383. doi: 10.1074/jbc.M701867200 PubMedCrossRefGoogle Scholar
  11. 11.
    Self AJ, Hall A (1995) Purification of recombinant Rho/Rac/G25K from Escherichia coli. Methods Enzymol 256:3–10PubMedCrossRefGoogle Scholar
  12. 12.
    Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A 85(23):8998–9002PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Paul A. Poland
    • 1
  • Carol L. Kinlough
    • 1
  • Rebecca P. Hughey
    • 1
    • 2
    Email author
  1. 1.Renal-Electrolyte Division, Department of Medicine, Laboratory of Epithelial Cell BiologyUniversity of PittsburghPittsburghUSA
  2. 2.School of MedicineUniversity of PittsburghPittsburghUSA

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