To obtain an easy and fast method for expression and analysis of in vitro mutated ORFa protein derivatives we have cloned and expressed the ORFa sequence in E. coli using the T7 expression system (Rosenberg et al., Gene 56:125, 1987). The yield of soluble ORFa protein in E. coli extracts was too low to detect DNA-binding in gel retardation experiments. However, substantial amounts of ORFa protein accumulate in the pellet fraction after cell breakage and low speed centrifugation. These insoluble aggregates are predominantly composed of recombinant protein. After solubilization in 6 M guanidinium chloride the ORFa extracts were renatured by dilution to relatively low concentration of the denaturant.
We began to study DNA-binding properties of renatured protein extract in gel retardation experiments. These experiments were performed with an Ac fragment containing 180 bp from the Ac 5' end and an unrelated pUC19 fragment. A protein-DNA complex was formed upon incubation of the renatured ORFa extract with the Ac 5' fragment. No complex was observed after incubation with the pUC19 fragment. In competition experiments we have shown that the renatured ORFa protein binds specifically to the 5' end of the Ac element as described for the soluble ORFa protein synthesized in cultured insect cells. However, the electrophoretic mobility of the DNA-protein complex is altered. We have also observed a nuclease activity in the renatured extracts which is not yet investigated in more detail.
In conclusion, we have demonstrated
that the DNA-binding activity of the ORFa protein synthesized in E.
coli can be reactivated after denaturation in guanidinium chloride.
to the MNL 64 On-Line Index
Return to the Maize Newsletter Index
Return to the Maize Genome Database Page