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 this study, we demonstrate the purification of a model protein, beta-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.
Desai
Urvee A
UA
Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA.
Sur
Gargi
G
Daunert
Sylvia
S
Babbitt
Ruth
R
Li
Qingshun
Q
D015153:Blotting, Western; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity; D004492:Edetic Acid / Q000494:pharmacology; D005821:Genetic Techniques; D005966:Glucuronidase / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012231:Rhizobium / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014026:Nicotiana / Q000821:virology
D002147:Calmodulin; D010940:Plant Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004492:Edetic Acid; D005966:Glucuronidase