Plasmid Construction and the Purification of Arabidopsis Plantancyaninand Lily Chemocyanin in a Prokaryotic System

Abstract

Chemocyanin is a chemotropic compound found in lily stigma that plays one of important factors in pollination. This basic copper binding protein, chemocyanin, functions in inducing pollen tube chemotropism. Also, its sequence shows similarity to plantacyanin, another copper binding protein of cell wall with unknown function. In order to purify proteins of lily chemocyain and Arabidopsis plantacyanin L1, L30, P1, and P30 werecontructed using PCR. L1, and L30contains lily chemocyanin domainand P1 and P30contains plantacyanin domain. L1 and P1 also contain signal peptides in addition to arabidopsis and plantacyanin domainswhile L30 and P32 do not contain that contains signal peptides. However, all four DNA contain NdeI and BamHI restriction sites. After transforming these to expression vector BL21, protein of P1 was purified using ultrafiltraion and protein of L30 was purified using sonication, 8M urea treatment, and ultrafiltration. It was found that the chemocyanin cannot be purified from intermembrane cytosol, however, plantacyanin can be purified.