Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells |
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Authors: | Mukesh Kumar Aruna K Behera Sanjay Kumar V R Srinivas Hasi R Das Avadhesha Surolia Rakha H Das |
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Institution: | (1) Genetic Engineering Division, Center For Biochemical Technology, Delhi University Campus, Mall Road, Delhi, 110 007, India;(2) Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, 560012, India;(3) Present address: Department of Internal Medicine, University of South Florida College of Medicine, 12901, Bruce B. Downs Boulevard, Tampa, FL 33612, USA |
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Abstract: | Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal 1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 × 106 cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) cross-reacted with re-PNA. The subunit molecular weight (30kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system. |
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Keywords: | Peanut agglutinin baculovirus insect cells |
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