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

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal beta 1-3GalNAc on the eukaryotic cell su rface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Auto grapha californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL13 93. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of th e polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expre sses 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 s erum-free medium. Intracellularly expressed protein (re-PNA) was purified t o apparent homogeneity by affinity chromatography using ECD-Sepharose. Poly clonal antibodies against natural PNA (n-PNA) crossreacted with re-PNA. The subunit molecular weight (30 kDa), hemagglutination activity, and carbohyd rate specificity of re-PNA were found to be identical to that of n-PNA, thu s confirming the abundant production of a functionally active protein in th e baculovirus expression system.