Purification of overexpressed gam gene protein from bacteriophage Mu by denaturation-renaturation techniques and a study of its DNA-binding properties.
Recombinant Mu gam gene protein (Mu GAM) synthesized in Escherichia coli accumulates in the form of insoluble inclusion bodies which, after cell lysis and low-speed centrifugation, can be recovered in the pellet fraction. This property was utilized in a purification procedure for Mu GAM based on guanidine hydrochloride denaturation-renaturation followed by a single DEAE-cellulose chromatographic step. The purified Mu GAM was shown by nitrocellulose-filter-binding experiments to bind with high affinity to linear double-stranded DNA and more weakly to supercoiled and single-stranded forms. Mu GAM protects linear DNA from degradation by a variety of exonucleases, but only weakly inhibits endonuclease activity. These results are in accord with a model of Mu GAM conferring protection from exonuclease activity by binding to the ends of DNA.
Abraham
Z H
ZH
School of Biological Sciences, University of Sussex, Brighton, U.K.
Symonds
N
N
D010583:Bacteriophage mu / Q000235:genetics* / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D003101:Collodion / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000037:antagonists & inhibitors; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors; D005092:Exonucleases / Q000037:antagonists & inhibitors; D005374:Filtration / Q000295:instrumentation; D015870:Gene Expression; D011489:Protein Denaturation; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C065410:gam protein, Coliphage; D003101:Collodion; D003851:Deoxyribonucleases; D005092:Exonucleases; D004262:DNA Restriction Enzymes