Ma. Jamela R. Revilleza, Alfred Galvez, Deanne C. Krenz,
Benito O. De Lumen, and Evelyn Mae T. Mendoza
Institute of Chemistry, University of the Philippines at Los Baños (UPLB)
Dept. of Nutritional Sciences, University of California, Berkeley
Institute of Plant Breeding, UPLB
http://doi.org/10.57043/transnastphl.1999.5773
Abstract
An 8 kD methionine-rich protein (MRP) (2D-1) was purified through a combination of selective enrichment of the albumin fraction through heparin-Sep ha rose affinity chromatography, 2D-SDS-PAGE and radiolabeling of methionine-containing proteins with [1-14C] iodoacetate. The MRP contains 8.6% methionine, 8.6% cysteine, 11.4% lysine and high amounts of glutamic and aspartic acids. The N-terminus sequence facilitated the synthesis of degenerate oligonucleotide primers for use in Polymerase Chain Reaction (PCR) to amplify cDNA fragments specifically coding for the gene. The PCR product was then used to screen a cDNA library and analyze the DNA sequence which confirmed the high sulfur amino content of 17.2% Southern analysis revealed the presence of two copies of the gene in the genome. This work lays down the groundwork for cloning of the gene which actually encodes the 8 kD MRP and a methionine poor 2.5 kD peptide .