Production of pure protein and antibodies and development of immunoassays to detect Ara h 3 levels in peanut varieties (1)
Peanut-allergic patients may react to doses of peanut as low as 10 mg, and complete avoidance can be difficult owing to cross-contamination or hidden sources of peanut products in foods.
Ara h 1 is a 63-kDa glycoprotein that associates into very stable trimers. Ara h 2 is a much smaller protein (17–20 kDa) that has been shown to have trypsin inhibitor activity. Ara h 3 is translated into a 60-kDa peptide, which is proteolyti- cally processed into 40- and 20-kDa subunits that associate into trimers and hexamers and has trypsin inhibitor activity. The 40-kDa subunit of Ara h 3, which houses the four dominant linear epitopes, has been optimised for the expression in E. coli and purified to near homogeneity.
Primers for PCR were designed that incorporated Nde I and Bam HI sites for cloning into pET9a. The primers used for PCR added the three N-terminal amino acid codons missing from the clone and terminated translation at codon 327. Double-stranded mutagenesis was subsequently applied to change four codons from those rarely used in E. coli to those that are highly used.
The 2M urea was found to release the highest amount, and subsequent removal of the urea did not result in the precipitation of the protein.
Most of the complex mixture of proteins do not bind the resin, and the Ara h 3 elutes as a nearly pure protein at approximately 400 mM NaCl.
Specific binding was observed to only the 40-kDa subunit of Ara h 3 both as purified protein and in the context of the mixture of proteins from whole peanuts.
The three isoforms of Ara h 3 are indicated with arrows. In this gel, the fastest migrating isoform appears to be missing or reduced in samples 194, 196, 199, 200 and 212. The slowest migrating isoform appears to be missing or reduced in samples 209, 210 and 213. At only 6% of the control, strain 24 has the lowest level of Ara h3 and the other stains with the exception of 35 accumulate between 20 and 60% of the control amount.
