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JBC, Vol. 254, Issue 19, 9565-9572, Oct, 1979
M. Bittner, R. L. Burke and B. M. Alberts
Detailed procedures are presented which allow reproducible preparation of
T4 gene 32 protein, a helix-destabilizing protein essential for DNA
replication and genetic recombination in T4 bacteriophage-infected
Escherichia coli cells. Although 32 protein can be purified to better than
99% homogeneity by any one of several procedures, these methods have been
developed to remove trace amounts of contaminating deoxyribonucleases,
which are present in high levels in the original infected cells. Two
alternative preparations are presented, each involving three
chromatographic steps. Both 32 proteins obtained are essentially
"nuclease-free," when tested at physiological salt concentrations. However,
we show here that the phenyl-Sepharose chromatography step, which is
necessary to remove an exonuclease activity active only at low salt
concentrations, also removes a second protein present in trace amounts. In
some cases, retention of this second protein is desirable, since it is
essential for obtaining RNA primed, de novo DNA chain starts in an in vitro
DNA replication system, when this system is constructed by mixing highly
purified preparations of each of the six replication proteins coded for by
T4 genes 32, 43, 44, 62, 45, and 41.
Purification of the T4 gene 32 protein free from detectable deoxyribonuclease activities
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