Isolation, purification, characterization of coconut (Cocos nucifera L.) globulins and the cloning of the cocos in cDNA

Cocosin was isolated and purified by salt extraction, Fast Protein Liquid Chromatography (FPLC)-gel filtration using HiLoad 26/60 Superdex 200 TM column and FPLC - anion exchange chromatography with RESOURCE QTM column. The native molecular weight of cocosin was estimated to be 326,000. Electrophoretic analysis revealed two closely migrating bands at approximately 34000 (acidic polypeptide) and another set of 2 bands at 24000 (basic polypeptide). Each set consisted of one darkly stained band and one lightly stained band. Three different isoforms of cocosin were identified after anion exchange chromatography. The N-terminal amino acid sequence of the 34 K band and 24 K band were SVRSVNEFRXE and GLEETQ, respectively. Using increasing concentrations of salt solution, cocosin was readily extracted by 0.35 M NaCl. In the absence of Beta-mercaptoethanol, the 55 K band representing the recombined submit species was darkly stained indicating the presence of disulfide linkage in the molecule. All the bands tested positively for the presence of carbohydrate moieties using periodic acid-Schiff's reagent. However, only the basic polypeptide stained positively for carbohydrate when con A- peroxidase was used. Cocosin comprised 80% of the coconut globulin. Gene-specific primers were designed by back translating the derived N-terminal amino acid sequence of cocosin. These were used to screen the coconut cDNA library for the cocosin gene. Of the 48 cDNA clones that were screened, nine showed a putative cocosin gene of 1.2 kb after colony polymerase chain reaction analysis. Amplification of pure plasmid DNA from four clones gave an 800 bp and a 400 bp PCR products. These DNA fragments were subjected to TOPO TA Cloning, however, only the 400 bp band gave clean DNA sequence for analysis. The basic cocosin amino acid sequence contained the conserved glycosylation site Nsub424-Asub425S426 and the highly conserved GLEET and ILRALP oligopeptides. A 37% necleotide homology and 14% amino acid homology with various 11S globulins were observed by Vector NTI sequence analysis. Low homology values were also observed with 7S (9%) and 8S globulins (14%). The 7S coconut globulin was isolated for the first time. The N-terminal amino acid sequence of 7S was EQEDPELQK. It showed a native molecular weight of 156000 and consisted of three polypeptides with molecular masses of 16000, 22000 and 24000. Twenty percent of the total globulins were accounted for by 7S globulin.