Expression of the catalytic subunit (UL54) and the accessory protein (UL44) of human cytomegalovirus DNA polymerase in a coupled in vitro transcription/translation system.
The catalytic subunit (UL54) and accessory protein (UL44) of human cytomegalovirus (HCMV) DNA polymerase have been cloned and expressed in an in vitro-coupled transcription/translation reticulocyte lysate system. The influence of the 5'-untranslated region (5'-UTR) on the efficiency of expression from the circular plasmids has been investigated. For expression of both UL54 and UL44, a truncated form of the alfalfa mosaic virus (AMV) RNA4 5'-UTR was found to be superior to the full-length AMV 5'-UTR or the original HCMV 5'-UTRs of different lengths. Protein products with Mr approximately 140 and 55 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in the UL54 and UL44 in vitro expression reactions, respectively. The properties of the expressed enzyme were compared with those of native HCMV DNA polymerase purified from HCMV-infected cells. DNA polymerase and 3'-5' exonuclease activities of the expressed UL54/UL44 complex were found to be dependent on salt concentration in the same manner as the activities of the native enzyme. The in vitro-expressed enzyme resembles the purified HCMV DNA polymerase in its affinity for deoxynucleoside triphosphates as well as in its sensitivity to known inhibitors (cidofovir diphosphate, ganciclovir triphosphate, and foscarnet). This straightforward method for protein expression may also be applicable to other enzymes where reproducible generation of fully functional products is desirable.
Cihlar
T
T
Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, USA. Tomas_Cihlar@gilead.com
Fuller
M D
MD
Cherrington
J M
JM
D017794:Alfalfa mosaic virus / Q000235:genetics; D001483:Base Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000201:enzymology* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D010957:Plasmids; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
D004268:DNA-Binding Proteins; C072217:ICP36 protein, Cytomegalovirus; D019384:Nucleic Acid Synthesis Inhibitors; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; C109367:UL54 protein, Human herpesvirus 5; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase