Gene 18 protein of bacteriophage T7. Overproduction, purification, and characterization.
Genetic and physical analyses indicate that gene 18 protein of bacteriophage T7 is essential for packaging of T7 DNA. T7 DNA is replicated via linear intermediates, culminating in the formation of concatemers many genomes in length which are then packaged into capsids. In infections with phage carrying amber mutations in gene 18, development is blocked at the concatemer stage. Biochemical studies on the role of gene 18 protein in concatemer processing and DNA packaging have been hampered by its low level of expression of gene 18 during T7 infections. We have cloned gene 18 on a plasmid downstream from the bacteriophage lambda PL promoter controlled by the temperature-sensitive lambda repressor encoded by c 1857. Thermal induction leads to the expression of the 10,000-Da gene 18 protein to the extent of approximately 10% of the total protein after 2 h. The overexpressed gene 18 protein is susceptible to proteolytic degradation, a condition that can be alleviated by expression in an Escherichia coli strain carrying the lon100 deletion which reduces production of protease La. Extracts of induced cells will complement an extract of T7-infected cells lacking gene 18 protein for packaging of exogenous T7 DNA. The assay has been used to monitor the purification of gene 18 protein to essential homogeneity. The identity of the purified protein has been confirmed by sequencing of the N terminus. Gel filtration analysis suggests that the native protein is an octomer. Treatment of gene 18 protein with 3 M guanidine hydrochloride denatures it to a monomer. Removal of the denaturing agent by dialysis regenerates the octomeric structure and the ability to complement packaging extracts.
D002850:Chromatography, Gel; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005814:Genes, Viral; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D011401:Promoter Regions, Genetic; D013604:T-Phages / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D014764:Viral Proteins; D004262:DNA Restriction Enzymes