Bacteriophage PRD1 terminal protein: expression of gene VIII in Escherichia coli and purification of the functional P8 product.
The gene VIII coding for the bacteriophage PRD1 terminal protein P8 has been cloned under the control of the lambda pL promoter. The recombinant plasmid thus obtained (pUSH20) was able to complement a mutation in the phage terminal-protein gene VIII. High expression of the cloned gene from this plasmid could be obtained by raising the growth temperature from 28 to 42 degrees C. This heat induction resulted in an increased synthesis of a protein of 30 kDa, the size expected for the P8 protein. When complemented with an extract of cells carrying the PRD1 DNA polymerase gene, the extract from the cells harboring the plasmid pUSH20 was able to form the P8-dGMP replication initiation complex. The PRD1 replication initiation reaction was optimized and used to detect the biological activity of the expressed terminal protein. Subsequently, P8 protein was purified to almost homogeneity and shown to be biologically functional after the various purification steps.
Savilahti
H
H
Department of Genetics, University of Helsinki, Finland.
Caldentey
J
J
Bamford
D H
DH
D001435:Bacteriophages / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005814:Genes, Viral; D005816:Genetic Complementation Test; D005838:Genotype; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D012486:Salmonella typhimurium / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification; D015678:Viral Structural Proteins / Q000235:genetics*
C054298:DNA terminal protein, Enterobacteria phage PRD1; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins