Bacteriophage T7 DNA polymerase: cloning and high-level expression.

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Phage T7 DNA polymerase is a complex of viral gene 5 protein and host thioredoxin.
• Cloning T7 gene 5 and adjacent genes into a plasmid allows for protein overproduction.

Purpose of the Study:

• To clone and overproduce phage T7 gene 5 protein and T7 DNA polymerase.
• To investigate the regulation of T7 gene 5 expression in Escherichia coli.

Main Methods:

• Cloning of T7 DNA fragment containing gene 5 into pBR322 plasmid.
• Transformation of thioredoxin-negative (trxA-) and thioredoxin-positive (trxA+) E. coli strains.
• Purification of T7 DNA polymerase using antithioredoxin immunoadsorbent chromatography.

Main Results:

• Significant overproduction of T7 gene 5 protein in trxA- E. coli.
• Identification of a potential E. coli RNA polymerase promoter upstream of T7 gene 5.
• Evidence suggesting autoregulation of T7 gene 5 expression by T7 DNA polymerase holoenzyme.
• Development of an efficient purification method for active T7 DNA polymerase.

Conclusions:

• The trxA- E. coli strain containing the recombinant plasmid is a valuable source for T7 gene 5 protein and T7 DNA polymerase.
• Autoregulation by the T7 DNA polymerase holoenzyme influences T7 gene 5 expression levels.