Cloning by genomic PCR and production of peanut agglutinin in Escherichia coli

Sharma, Vivek and Surolia, Avadhesha (1994) Cloning by genomic PCR and production of peanut agglutinin in Escherichia coli. In: Gene, 148 (2). pp. 299-304.

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Abstract

Using the polymerase chain reaction, the coding sequence for peanut agglutinin (PNA) was cloned and expressed in Escherichia coli. Amplified PNA is identical to previously reported cDNA, suggesting the absence of any introns in PNA gene. Recombinant (re-) PNA forms inclusion bodies in E. coli. Production of PNA was confirmed by probing Western blots with polyclonal anti-PNA immunoglobulin G. Inclusion bodies were solubilized with 6 M guanidine-HCl and renatured by rapid dilution in the presence of metal ions. The renatured lectin was then purified by affinity chromatography. The re-lectin shows carbohydrate-binding properties similar to the natural PNA. This expression system provides a model for future mutagenesis studies of the carbohydrate-binding site and thus facilitates ongoing efforts to explore the molecular basis for the specificity of lectin-carbohydrate interaction.

Item Type:	Journal Article
Publication:	Gene
Publisher:	Elsevier Science
Additional Information:	Copyright of this article belongs to Elsevier Science.
Keywords:	Arachis hypogea; legume lectin; polymerase chain reaction; inclusion bodies; haemagglutination
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	21 May 2010 06:03
Last Modified:	19 Sep 2010 06:01
URI:	http://eprints.iisc.ac.in/id/eprint/27503

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