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Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells

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Bioscience Reports

Abstract

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Galβ1-3GalNAc 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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Author notes

  1. Aruna K. Behera

    Present address: Department of Internal Medicine, University of South Florida College of Medicine, 12901, Bruce B. Downs Boulevard, Tampa, FL, 33612, USA

Authors and Affiliations

  1. Genetic Engineering Division, Center For Biochemical Technology, Delhi University Campus, Mall Road, Delhi, 110 007, India

    Aruna K. Behera, Sanjay Kumar & Rakha H. Das

  2. Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, 560012, India

    V. R. Srinivas & Avadhesha Surolia

  3. Department of Internal Medicine, University of South Florida College of Medicine, 12901, Bruce B. Downs Boulevard, Tampa, FL, 33612, USA

    Mukesh Kumar

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  1. Mukesh Kumar
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Kumar, M., Behera, A.K., Kumar, S. et al. Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells. Biosci Rep 19, 227–234 (1999). https://doi.org/10.1023/A:1020234005099

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