Overexpression, purification, and analysis of the c1 repressor protein of Pseudomonas aeruginosa bacteriophage D3

• Published in: Canadian journal of microbiology Vol. 43; no. 3; p. 220
• Main Authors: Farinha, M A, Kropinski, A M
• Format: Journal Article
• Language: English
• Published: Canada 01.03.1997
• Subjects: Amino Acid Sequence; Bacteriophages - genetics; Base Sequence; Cloning, Molecular; DNA Footprinting; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Open Reading Frames; Operator Regions, Genetic; Plasmids; Pseudomonas aeruginosa - virology; Repressor Proteins - genetics; Repressor Proteins - physiology; RNA, Messenger - analysis; RNA, Messenger - metabolism; Sequence Analysis, DNA; Transcription, Genetic; Viral Proteins; Viral Regulatory and Accessory Proteins
• ISSN: 0008-4166
• DOI: 10.1139/m97-030

A 3.1-kb region of the bacteriophage D3 genome which contains the immunity functions has recently been sequenced (GenBank accession No. L22692). Sequence analysis indicated the presence of a putative repressor gene (c1) whose protein product functions to maintain the bacteriophage genome as a stably integrated prophage in the chromosome of Pseudomonas aeruginosa. A plasmid was constructed that overexpresses repressor C1 protein under control of P(tac) in Escherichia coli. C1 protein was subsequently purified and characterized as a 223 amino acid protein with specific binding affinity for 14-base imperfect palindromic operator sequences located on the genome of bacteriophage D3. N-terminal protein sequence data obtained from automated Edman degradation (16 cycles) of purified repressor protein were identical to the predicted sequence based on DNA sequence analysis of the c1 open reading frame.