Cloning and overexpression of the gene encoding bacteriophage T5 DNA polymerase

• Published in: Gene Vol. 97; no. 1; pp. 13 - 19
• Main Authors: Chatterjeea, Deb K., Fujimurab, Robert K., Campbella, James H., Gerarda, Gary F.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 1991; Amsterdam Elsevier; New York, NY
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; Cloning, Molecular; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; Escherichia coli - genetics; exonuclease III deletion; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes, Viral; Genes. Genome; Genetic Vectors; insoluble protein; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; nucleotide sequence; Promoter Regions, Genetic; Recombinant DNA; Restriction Mapping; Sequence Homology, Nucleic Acid; T-Phages - enzymology; T-Phages - genetics; T5 phage promoter; thioredoxin; Thioredoxins - pharmacology; Transcription, Genetic; Viral Structural Proteins - genetics; ORF; RT; SDS; exonuclease III deletion; thioredoxin; DTT; M-MLV; nt; EtdBr; T5 phage promoter; AMV; PAGE; Recombinant DNA; bp; Ap; nucleotide sequence; tsp; PA; T5Pol; kb; Pollk; IPTG; insoluble protein; Trx; DNA polymerase; Upstream sequence; Nucleotide sequence; Enzyme; Transcription promoter; Gene expression; Virus; Styloviridae; DNA; Replication; Phage T5; Molecular cloning; Phage
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(91)90004-U

T5 DNA polymerase (T5Pol), an essential enzyme for bacteriophage T5 DNA replication, is unusual because of its high processivity and strand-displacing ability. These two properties in a single polypeptide make T5Pol an ideal candidate for structural and functional analysis. Therefore, the structural gene encoding the DNA polymerase of bacteriophage T5 ( T5pol) has been cloned and overexpressed in Escherichia coli. Elimination of sequences upstream from the 5' end of the T5pol by exonuclease III digestion was necessary to obtain stable clones containing a full-length structural gene. Determination of the nucleotide (nt) sequence of the region deleted during clone construction revealed the presence of a promoter sequence having extensive homology with known T5 phage 'early' promoters. By primer extension ofmRNA isolated from T5 phage-infected cells, two successive G residues located 6 and 7 nt downstream from the − 10 region of this promoter were identified as the initiating nt at the 5' end of T5pol mRNA. T5Pol produced in E. coli from the cloned gene under control of a tac or phage λp l promoter represented as much as 40% of total cell protein. The majority of the T5Pol present in extracts of E. coli was insoluble. The amount of active enzyme present was estimated to be a maximum of tenfold higher than that found in extracts of T5 phage-infected cells.