Cloning by genomic PCR and production of peanut agglutinin in Escherichia coli.
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.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
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Surolia
D010367:Arachis; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D037102:Lectins / Q000235:genetics*; D008969:Molecular Sequence Data; D019887:Peanut Agglutinin; D037121:Plant Lectins; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics
D017931:DNA Primers; D004274:DNA, Recombinant; D037102:Lectins; D019887:Peanut Agglutinin; D037121:Plant Lectins; D011994:Recombinant Proteins