EXPRESSION OF SUNFLOWER HOMEODOMAIN CONTAINING PROTEINS IN ESCHERICHIA-COLI - PURIFICATION AND FUNCTIONAL-STUDIES

Complementary DNA sequences encoding different portions of two sunflow er homeodomain proteins were cloned in-frame in the expression vectors pRSET and pGEX-3X. When introduced into competent Escherichia coli ce lls and induced, the resulting plasmids directed the expression of lar ge amounts (5-10% of total cellular protein) of the encoded polypeptid es, As a rule, fusions in pRSET rendered insoluble proteins, while fus ions in pGEX were soluble and could be purified in a single step by se lective absorption onto glutathione-agarose beads, followed by elution with free glutathione. The purified proteins showed both glutathione S-transferase and DNA-binding activity, indicating that they retain th eir native conformation. The expression-purification protocol that was employed allowed the isolation of up to 0.7 mg of protein per gram of transformed cells. One of the fusion proteins, RH11 (which is a fusio n of the homeodomain protein HAHR1 in pRSET), though insoluble, was ab le to bind DNA when spotted onto a nitrocellulose filter. This protein could also be simply purified in large amounts by electroelution from sodium dodecyl sulfate-polyacrylamide gels and used to elicit antibod ies which recognized both the transgenic fusion and the native protein from sunflower nuclei. Our results clearly show that vector choice is a critical parameter for obtaining large amounts of a desired protein for particular purposes. (C) 1998 Academic Press.