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

Kumar, Mukesh and Behera, Aruna K and Kumar, Sanjay and Srinivas, VR and Das, Hasi R and Surolia, Avadhesha and Das, Rakha H (1999) Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells. In: Bioscience Reports, 19 (3). pp. 227-234.

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Abstract

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal beta 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 x 10(6) 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) crossreacted with re-PNA. The subunit molecular weight (30 kDa), 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.

Item Type:	Journal Article
Publication:	Bioscience Reports
Publisher:	Springer
Additional Information:	Copyright of this article belongs to Springer.
Keywords:	Peanut agglutinin;Baculovirus;Insect cells.
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	19 Jul 2009 07:03
Last Modified:	19 Sep 2010 05:27
URI:	http://eprints.iisc.ac.in/id/eprint/19334

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