Cloning, expression, and biochemical characterization of hexahistidine-tagged terminase proteins.
The terminase enzyme from bacteriophage lambda is composed of two viral proteins (gpA, 73.2 kDa; gpNu1, 20.4 kDa) and is responsible for packaging viral DNA into the confines of an empty procapsid. We are interested in the genetic, biochemical, and biophysical properties of DNA packaging in phage lambda and, in particular, the nucleoprotein complexes involved in these processes. These studies require the routine purification of large quantities of wild-type and mutant proteins in order to probe the molecular mechanism of DNA packaging. Toward this end, we have constructed a hexahistidine (hexa-His)-tagged terminase holoenzyme as well as hexa-His-tagged gpNu1 and gpA subunits. We present a simple, one-step purification scheme for the purification of large quantities of the holoenzyme and the individual subunits directly from the crude cell lysate. Importantly, we have developed a method to purify the highly insoluble gpNu1 subunit from inclusion bodies in a single step. Hexa-His terminase holoenzyme is functional in vivo and possesses steady-state and single-turnover ATPase activity that is indistinguishable from wild-type enzyme. The nuclease activity of the modified holoenzyme is near wild type, but the reaction exhibits a greater dependence on Escherichia coli integration host factor, a result that is mirrored in vivo. These results suggest that the hexa-His-tagged holoenzyme possesses a mild DNA-binding defect that is masked, at least in part, by integration host factor. The mild defect in hexa-His terminase holoenzyme is more significant in the isolated gpA-hexa-His subunit that does not appear to bind DNA. Moreover, whereas the hexa-His-tagged gpNu1 subunit may be reconstituted into a holoenzyme complex with wild-type catalytic activities, gpA-hexa-His is impaired in its interactions with the gpNu1 subunit of the enzyme. The results reported here underscore that a complete biochemical characterization of the effects of purification tags on enzyme function must be performed prior to their use in mechanistic studies.
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Molecular Biology Program, University of Iowa, Iowa City, Iowa 52242, USA.
Woods
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Feiss
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Catalano
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D010582:Bacteriophage lambda / Q000201:enzymology*; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000235:genetics*; D004720:Endonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D006639:Histidine / Q000235:genetics; D034982:Integration Host Factors; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D019065:Virus Assembly / Q000235:genetics
D001426:Bacterial Proteins; D004279:DNA, Viral; D034982:Integration Host Factors; D011994:Recombinant Proteins; D014764:Viral Proteins; C050856:lambda phage protein Nu 1; D006639:Histidine; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C030894:terminase; D000251:Adenosine Triphosphatases