Expression of sunflower homeodomain containing proteins in Escherichia coli: purification and functional studies

Protein Expression and Purification
C M PalenaRaquel Chan

Abstract

Complementary DNA sequences encoding different portions of two sunflower homeodomain proteins were cloned in-frame in the expression vectors pRSET and pGEX-3X. When introduced into competent Escherichia coli cells and induced, the resulting plasmids directed the expression of large amounts (5-10% of total cellular protein) of the encoded polypeptides. As a rule, fusions in pRSET rendered insoluble proteins, while fusions in pGEX were soluble and could be purified in a single step by selective 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 their 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 fusion of the homeodomain protein HAHR1 in pRSET), though insoluble, was able 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 antibodies which recognized both the transgenic fusion and the n...Continue Reading

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Citations

Jun 17, 2011·Journal of Experimental Botany·Eduardo F MufarregeGraciela C Curi
Oct 8, 2004·Journal of Biochemistry and Molecular Biology·María Eugenia ZanettiClaudia A Casalongué
Jul 3, 2002·The Journal of Biological Chemistry·Adriana E TronDaniel H Gonzalez
May 10, 2015·Plant Molecular Biology·Jesica RaineriRaquel Lia Chan
Sep 29, 2007·Archives of Biochemistry and Biophysics·Raúl N Comelli, Daniel H Gonzalez
May 25, 2010·Developmental Cell·Wolfgang BuschJan U Lohmann

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