Expression, purification and characterization of peanut (Arachis hypogaea) agglutinin (PNA) from baculovirus infected insect cells.

Abstract:

:Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Galbeta1-3GalNAc 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) cross-reacted 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.

journal_name

Biosci Rep

journal_title

Bioscience reports

authors

Kumar M,Behera AK,Kumar S,Srinivas VR,Das HR,Surolia A,Das RH

doi

10.1023/a:1020234005099

keywords:

subject

Has Abstract

pub_date

1999-06-01 00:00:00

pages

227-34

issue

3

eissn

0144-8463

issn

1573-4935

journal_volume

19

pub_type

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