Figure 3.

Purification and Analysis of Wild-Type and eto2 ACS5 Proteins.

(A) SDS-PAGE analysis of the purification of ACS5^WT. E. coli cells harboring the IMPACT-ACS5^WT expression construct were induced, and the protein was extracted and applied to a chitin agarose column as described in Methods. Various extracts were analyzed by SDS-PAGE, and the proteins were visualized by Coomassie blue staining. Lane 1, crude extract; lane 2, column flow through; lane 3, column wash; lane 4, first fraction after the activation of intein protease; lane 5, second fraction after the activation of intein protease. The positions of migration of the molecular mass markers are indicated at left.

(B) SDS-PAGE analysis of the purification of ACS5^eto2. Lanes are as in (A).

(C) Specific activity of purified recombinant ACS5 proteins. The fractions analyzed in lanes 5 from (A) and (B) were assayed for ACS activity as described in Methods. The activity was normalized to the amount of protein present. Values shown are means of three assays ± sd. The inset shows an immunoblot of proteins used in the assay probed with an anti-ACS5 antibody. WT, wild type.