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), 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.
Kumar
M
M
Genetic Engineering Division, Center for Biochemical Technology, Delhi University Campus, India. dasrh@hotmail.com
Behera
A K
AK
Kumar
S
S
Srinivas
V R
VR
Das
H R
HR
Surolia
A
A
Das
R H
RH
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors; D006384:Hemagglutination; D006801:Humans; D015151:Immunoblotting; D019887:Peanut Agglutinin / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D018411:Spodoptera
D019887:Peanut Agglutinin