, Volume 235, Issue 4, pp 863–871 | Cite as

Expression of the affinity tags, glutathione-S-transferase and maltose-binding protein, in tobacco chloroplasts

  • Niaz Ahmad
  • Franck Michoux
  • James McCarthy
  • Peter J. Nixon
Emerging Technologies


Chloroplast transformation offers an exciting platform for the safe, inexpensive and large-scale production of recombinant proteins in plants. An important advantage for the isolation of proteins produced in the chloroplast would be the use of affinity tags for rapid purification by affinity chromatography. To date, only His-tags have been used. In this study, we have tested the feasibility of expressing two additional affinity tags: glutathione-S-transferase (GST) and a His-tagged derivative of the maltose-binding protein (His6-MBP). By using the chloroplast 16S rRNA promoter and 5′ untranslated region of phage T7 gene 10, GST and His6-MBP were expressed in homoplastomic tobacco plants at approximately 7% and 37% of total soluble protein, respectively. GST could be purified by one-step-affinity purification using a glutathione column. Much better recoveries were obtained for His6-MBP by using a twin-affinity purification procedure involving first immobilised nickel followed by binding to amylose. Interestingly, expression of GST led to cytoplasmic male sterility. Overall, our work expands the tools available for purifying recombinant proteins from the chloroplast.


Affinity tags Chloroplast transformation Cytoplasmic male sterility Glutathione-S-transferase Maltose-binding protein 



Cytoplasmic male sterility






Immobilised metal affinity chromatography


Maltose-binding protein




Protein extraction buffer


Total soluble protein


Untranslated region


  1. Bilang J, Macdonald H, King PJ, Sturm A (1993) A soluble auxin-binding protein from Hyoscyamus muticus is a glutathione S-transferase. Plant Physiol 102:29–34PubMedCrossRefGoogle Scholar
  2. Birky CW Jr (1995) Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolution. Proc Natl Acad Sci USA 92:11331–11338PubMedCrossRefGoogle Scholar
  3. Bock R, Warzecha H (2010) Solar-powered factories for new vaccines and antibiotics. Trends Biotechnol 28:246–252PubMedCrossRefGoogle Scholar
  4. Boehm M, Nield J, Zhang P, Aro EM, Komenda J, Nixon PJ (2009) Structural and mutational analysis of band 7 proteins in the cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol 191:6425–6435PubMedCrossRefGoogle Scholar
  5. Cecchetti V, Pomponi M, Altamura MM, Pezzotti M, Marsilio S, DíAngeli S, Tornielli GB, Costantino P, Cardarelli M (2004) Expression of rolB in tobacco flowers affects the coordinated processes of anther dehiscence and style elongation. Plant J 38:512–525PubMedCrossRefGoogle Scholar
  6. Cecchetti V, Altamura MM, Falasca G, Costantino P, Cardarelli M (2008) Auxin regulates Arabidopsis anther dehiscence, pollen maturation, and filament elongation. Plant Cell 20:1760–1774PubMedCrossRefGoogle Scholar
  7. Chakauya E, Chikwamba R, Rybicki EP (2006) Riding the tide of biopharming win Africa: considerations for risk assessment. S Afr J Sci 102:284–288Google Scholar
  8. Cheng Y, Dai X, Zhao Y (2006) Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis. Genes Dev 20:1790–1799PubMedCrossRefGoogle Scholar
  9. Daniell H, Khan MS, Allison L (2002) Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci 7:84–91PubMedCrossRefGoogle Scholar
  10. Douglas KT (1987) Mechanism of action of glutathione dependent enzymes. Adv Enzymol Relat Areas Mol Biol 59:103–167PubMedGoogle Scholar
  11. Dove A (2002) Uncorking the biomanufacturing bottleneck. Nat Biotechnol 20:777–779PubMedCrossRefGoogle Scholar
  12. Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7:152–158PubMedCrossRefGoogle Scholar
  13. Girish V, Vijayalakshmi A (2004) Affordable image analysis using NIH Image/ImageJ. Indian J Cancer 41:47PubMedGoogle Scholar
  14. Kapust RB, Waugh DS (1999) Escherichia coli maltose binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8:1668–1674PubMedCrossRefGoogle Scholar
  15. Koya V, Moayeri M, Leppla SH, Daniell H (2005) Plant-based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge. Infect Immun 73:8266–8274PubMedCrossRefGoogle Scholar
  16. Kuroda H, Maliga P (2001) Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs. Nucleic Acids Res 29:970–975PubMedCrossRefGoogle Scholar
  17. Kusnadi AR, Nikolov ZL, Howard JA (1997) Production of recombinant proteins in transgenic plants: practical considerations. Biotechnol Bioeng 56:473–484PubMedCrossRefGoogle Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  19. Le Martret B, Poage M, Shiel K, Nugent GD, Dix PJ (2011) Tobacco chloroplast transformants expressing genes encoding dehydroascorbate reductase, glutathione reductase, and glutathione-S-transferase, exhibit altered anti oxidant metabolism and improved abiotic stress tolerance. Plant Biotech J 9(66):1–673Google Scholar
  20. Leelavathi S, Reddy VS (2003) Chloroplast expression of His-tagged GUS-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors. Mol Breed 11:49–58CrossRefGoogle Scholar
  21. Lössl AG, Waheed MT (2011) Chloroplast-derived vaccines against human diseases: achievements, challenges and scopes. Plant Biotech J 9:527–539CrossRefGoogle Scholar
  22. Lossl A, Eibl C, Harloff HJ, Jung C, Koop HU (2003) Polyester synthesis in transplastomic tobacco (Nicotiana tabacum L.): significant contents of polyhydroxybutyrate are associated with growth reduction. Plant Cell Rep 21:891–899PubMedGoogle Scholar
  23. Maliga P (2002) Engineering the plastid genome of higher plants. Curr Opin Plant Biol 5:164–172PubMedCrossRefGoogle Scholar
  24. Maliga P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol 21:20–28PubMedCrossRefGoogle Scholar
  25. Maliga P, Bock R (2011) Plastid biotechnology: food, fuel, and medicine for the 21st century. Plant Physiol 155:1501–1510PubMedCrossRefGoogle Scholar
  26. Michoux F (2008) Developing new strategies for the production of foreign proteins in higher plant chloroplast. PhD thesis. Imperial College, LondonGoogle Scholar
  27. Michoux F, Ahmad N, McCarthy J, Nixon PJ (2011) Contained and high-level production of recombinant protein in plant chloroplasts using a temporary immersion bioreactor. Plant Biotechnol J 9:575–584PubMedCrossRefGoogle Scholar
  28. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 79:197–205Google Scholar
  29. Nallamsetty S, Austin BP, Penrose KJ, Waugh DS (2005) Gateway vectors for the production of combinatorially-tagged His6-MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli. Protein Sci 14:2964–2971PubMedCrossRefGoogle Scholar
  30. Niittyla T, Messerli G, Trevisan M, Chen J, Smith AM, Zeeman SC (2004) A previously unknown maltose transporter essential for starch degradation in leaves. Science 303:87–89PubMedCrossRefGoogle Scholar
  31. Oey M, Lohse M, Kreikemeyer B, Bock R (2009a) Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. Plant J 57:436–445PubMedCrossRefGoogle Scholar
  32. Oey M, Lohse M, Scharff LB, Kreikemeyer B, Bock R (2009b) Plastid production of protein antibiotics against pneumonia via a new strategy for high-level expression of antimicrobial proteins. Proc Natl Acad Sci USA 106:6579–6584PubMedCrossRefGoogle Scholar
  33. Petersen K, Bock R (2011) High-level expression of a suite of thermostable cell wall-degrading enzymes from the chloroplast genome. Plant Mol Biol 76:311–321PubMedCrossRefGoogle Scholar
  34. Ruhlman T, Verma D, Samson N, Daniell H (2010) The role of heterologous chloroplast sequence elements in transgene integration and expression. Plant Physiol 152:2088–2104PubMedCrossRefGoogle Scholar
  35. Ruiz ON, Daniell H (2005) Engineering cytoplasmic male sterility via the chloroplast genome by expression of β-ketothiolase. Plant Physiol 138:1232–1246PubMedCrossRefGoogle Scholar
  36. Smith DB, Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31–40PubMedCrossRefGoogle Scholar
  37. Spreitzer RJ, Salvucci ME (2002) Rubisco: structure, regulatory interactions, and possibilities for a better enzyme. Annu Rev Plant Biol 53:449–475PubMedCrossRefGoogle Scholar
  38. Staub JM, Garcia B, Graves J, Hajdukiewicz PT, Hunter P, Nehra N, Paradkar V, Schlittler M, Carroll JA, Spatola L, Ward D, Ye G, Russell DA (2000) High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol 18:333–338PubMedCrossRefGoogle Scholar
  39. Sticklen MB (2008) Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol. Nat Rev Genet 9:433–443PubMedCrossRefGoogle Scholar
  40. Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913–917PubMedCrossRefGoogle Scholar
  41. Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87:8526–8530PubMedCrossRefGoogle Scholar
  42. Wachter A, Wolf S, Steininger H, Bogs J, Rausch T (2005) Differential targeting of GSH1 and GSH2 is achieved by multiple transcription initiation: implications for the compartmentation of glutathione biosynthesis in the Brassicaceae. Plant J 41:15–30PubMedCrossRefGoogle Scholar
  43. Wagner U, Edwards R, Dixon DP, Mauch F (2002) Probing the diversity of the Arabidopsis glutathione S-transferase gene family. Plant Mol Biol 49:515–532PubMedCrossRefGoogle Scholar
  44. Watson J, Koya V, Leppa S, Daniell H (2004) Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop. Vaccine 22:4374–4384PubMedCrossRefGoogle Scholar
  45. Zechmann B, Koffler BE, Russell SD (2011) Glutathione synthesis is essential for pollen germination in vitro. BMC Plant Biol 11:54–65PubMedCrossRefGoogle Scholar
  46. Zettl R, Schell J, Palme K (1994) Photoaffinity labeling of Arabidopsis thaliana plasma membrane vesicles by 5-azido-[7-3H] indole-3-acetic acid: identification of a glutathione S-transferase. Proc Natl Acad Sci USA 91:689–693PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Niaz Ahmad
    • 1
  • Franck Michoux
    • 1
  • James McCarthy
    • 2
  • Peter J. Nixon
    • 1
  1. 1.Division of Molecular Biosciences, Wolfson Biochemistry BuildingImperial College LondonLondonUK
  2. 2.Centre de Recherche NestléToursFrance

Personalised recommendations