Cloning, expression and in vitro characterisation of the M13 gene 5 protein.
The gene 5 protein encoded in the genome of bacteriophage M13 is a single stranded DNA binding protein essential for phage replication. We have cloned a fragment of the M13 genome containing gene 5, and investigated the effect of upstream elements on expression of the gene by means of Bal 31 deletion analysis. The gene was also expressed from the lac promoter of the phagemid vector pUC119, and the recombinant protein purified and characterised for DNA binding. The affinity of the recombinant protein for single-stranded DNA was shown to be essentially identical to that of wild type gene 5 protein. Wild type gene 5 protein has a glutamic acid residue at position 30 which, on the basis of the crystal structure, was believed to play a role in maintaining the tertiary structure of the protein through the formation of a salt bridge with arginine-80. We show that substitution of glutamic acid at position 30 by lysine does not impair DNA binding, suggesting that a salt bridge between glutamate-30 and arginine-80 is not essential for the structural integrity of the gene 5 protein as previously proposed.
Biophysics Laboratories, University of Portsmouth, UK.
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Turner
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Kneale
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004706:Endodeoxyribonucleases; D017353:Gene Deletion; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012045:Regulatory Sequences, Nucleic Acid; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C057816:gene 5 protein, Enterobacteria phage M13; D004706:Endodeoxyribonucleases; C038407:exonuclease Bal 31