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We have cloned and expressed the yeast DNA helicase A in Escherichia coli at a high level (∼30 mg/L of culture) in soluble form. We describe here a simple two-step purification protocol that produces reasonable quantities of homogeneous enzyme. In denaturing gel electrophoresis the enzyme behaved as a ∼90 kDa protein. The native structure, determined by gel-filtration studies, appeared to be hexameric and its quaternary structure was salt (NaCl) dependent. In low-salt buffers (containing 50 mM NaCl), the enzyme eluted in a single activity peak at an elution volume that appeared to correlate with a possible hexameric structure. In higher salt buffer (containing greater than 150 mM NaCl), the enzyme eluted as smaller assemblies (monomer/dimer). The recombinant helicase A was able to hydrolyze ATP or dATP with equal efficiency. The ATPase activity of the enzyme was absolutely DNA-dependent. The …