Expression of the catalytic subunit (UL54) and the accessory protein (UL44) of human cytomegalovirus DNA polymerase in a coupled in vitro transcription/translation system

被引:35
作者
Cihlar, T
Fuller, MD
Cherrington, JM
机构
[1] Gilead Sciences, Foster City, CA 94404
关键词
D O I
10.1006/prep.1997.0781
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 M-r approximate to 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. (C) 1997 Academic Press.
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页码:209 / 218
页数:10
相关论文
共 30 条
[1]   A CONSERVED 3'-]5' EXONUCLEASE ACTIVE-SITE IN PROKARYOTIC AND EUKARYOTIC DNA-POLYMERASES [J].
BERNAD, A ;
BLANCO, L ;
LAZARO, JM ;
MARTIN, G ;
SALAS, M .
CELL, 1989, 59 (01) :219-228
[2]  
BRITT WJ, 1996, FIELDS VIROLOGY, P2493
[3]   KINETIC-ANALYSIS OF THE INTERACTION BETWEEN THE DIPHOSPHATE OF (S)-1-(3-HYDROXY-2-PHOSPHONYLMETHOXYPROPYL) CYTOSINE, DDCTP, AZTTP, AND FIAUTP WITH HUMAN DNA POLYMERASE-BETA AND POLYMERASE-GAMMA [J].
CHERRINGTON, JM ;
ALLEN, SJ ;
MCKEE, BH ;
CHEN, MS .
BIOCHEMICAL PHARMACOLOGY, 1994, 48 (10) :1986-1988
[4]  
COEN DM, 1996, DNA REPLICATION EUKA, P495
[5]  
CRUTE JJ, 1989, J BIOL CHEM, V264, P19266
[6]   EXPRESSION OF HERPES-SIMPLEX VIRUS TYPE-1 DNA-POLYMERASE GENE BY INVITRO TRANSLATION AND EFFECTS OF GENE DELETIONS ON ACTIVITY [J].
DORSKY, DI ;
CRUMPACKER, CS .
JOURNAL OF VIROLOGY, 1988, 62 (09) :3224-3232
[7]   HIGH-LEVEL EXPRESSION OF DNA-POLYMERASES FROM HERPESVIRUSES [J].
ERTL, PF ;
THOMAS, MS ;
POWELL, KL .
JOURNAL OF GENERAL VIROLOGY, 1991, 72 :1729-1734
[8]   PHYSICAL AND FUNCTIONAL INTERACTION OF HUMAN CYTOMEGALOVIRUS DNA-POLYMERASE AND ITS ACCESSORY PROTEIN (ICP36) EXPRESSED IN INSECT CELLS [J].
ERTL, PF ;
POWELL, KL .
JOURNAL OF VIROLOGY, 1992, 66 (07) :4126-4133
[9]   BOTH THE 5' UNTRANSLATED REGION AND THE SEQUENCES SURROUNDING THE START SITE CONTRIBUTE TO EFFICIENT INITIATION OF TRANSLATION INVITRO [J].
FALCONE, D ;
ANDREWS, DW .
MOLECULAR AND CELLULAR BIOLOGY, 1991, 11 (05) :2656-2664
[10]  
GLASS CA, 1990, PROMEGA NOTES, V26, P6