Planta

, 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

Abstract

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.

Keywords

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

Abbreviations

CMS

Cytoplasmic male sterility

DIG

Digoxigenin

GST

Glutathione-S-transferase

IMAC

Immobilised metal affinity chromatography

MBP

Maltose-binding protein

Ni

Nickel

PEB

Protein extraction buffer

TSP

Total soluble protein

UTR

Untranslated region

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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

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