SDS–gel electrophoretic
patterns of GST–chHAT-1 fusion protein-containing fractions
at different purification steps, and substrate specificity of the recombinant
chHAT-1. (A) Protein samples prepared were subjected
to 10% SDS–PAGE, followed by Coomassie brilliant
blue staining. Lane 1, whole cell lysate of BL-21 cells containing
the pGEX-2TKchHAT-1 plasmid without induction by IPTG; lane 2, lysate
of BL-21 cells containing the pGEX-2TKchHAT-1 plasmid with induction
by 50 µM IPTG; lane 3, complex beads
containing chHAT-1; lane 4, chHAT-1 fraction purified with glutathione–Sepharose
beads. The standard molecular weights are indicated. GST–HAT-1,
GST–chHAT-1 fusion protein. (B) The in vitro acetylation reaction mixture (described below) was subjected to 15% SDS–PAGE, fixed, stained
with Coomassie brilliant blue (a) and then exposed to an imaging
plate (b). Lane 1, recombinant chicken GCN5 (our unpublished data); lane
2, recombinant chHAT-1; lane 3, without the enzyme as a negative
control, using chicken histones prepared from DT40 cells as substrates.