Expression and Affinity Purification of Recombinant Proteins from Plants

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In...

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Veröffentlicht in:Protein expression and purification 2002-06, Vol.25 (1), p.195-202
Hauptverfasser: Desai, Urvee A., Sur, Gargi, Daunert, Sylvia, Babbitt, Ruth, Li, Qingshun
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container_end_page 202
container_issue 1
container_start_page 195
container_title Protein expression and purification
container_volume 25
creator Desai, Urvee A.
Sur, Gargi
Daunert, Sylvia
Babbitt, Ruth
Li, Qingshun
description With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, β-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system.
doi_str_mv 10.1006/prep.2002.1627
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subjects affinity tag
Blotting, Western
calmodulin
Calmodulin - chemistry
Calmodulin - metabolism
Chromatography, Affinity
Edetic Acid - pharmacology
expression
Genetic Techniques
Glucuronidase - metabolism
isolation
Life Sciences (General)
Nicotiana - virology
Plant Proteins - chemistry
Plant Proteins - isolation & purification
Plants, Genetically Modified
Plasmids - metabolism
Protein Binding
Protein Conformation
Recombinant Fusion Proteins - metabolism
Recombinant Proteins - chemistry
Recombinant Proteins - isolation & purification
Rhizobium - metabolism
RNA, Viral - genetics
Spectrometry, Fluorescence
transgenic plants
title Expression and Affinity Purification of Recombinant Proteins from Plants
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