Related Experiment Videos
Purification of overexpressed gam gene protein from bacteriophage Mu by denaturation-renaturation techniques and a
1School of Biological Sciences, University of Sussex, Brighton, U.K.
Abstract:
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.
Insights
Recombinant Mu gam gene protein (Mu GAM) can be purified from Escherichia coli inclusion bodies. Purified Mu GAM binds linear DNA and protects it from exonuclease degradation, suggesting it binds DNA ends.
Area of Science:
- Molecular Biology
- Protein Biochemistry
- Genetics
Background:
- Recombinant Mu gam gene protein (Mu GAM) is expressed in Escherichia coli.
- Mu GAM forms insoluble inclusion bodies, facilitating initial recovery.
- Understanding Mu GAM's DNA binding and protective properties is crucial.
Purpose of the Study:
- To develop a purification strategy for recombinant Mu GAM.
- To characterize the DNA binding affinity and specificity of Mu GAM.
- To investigate Mu GAM's role in protecting DNA from enzymatic degradation.
Main Methods:
- Protein expression in Escherichia coli and isolation of inclusion bodies.
- Denaturation-renaturation using guanidine hydrochloride.
- Purification via DEAE-cellulose chromatography.
- Nitrocellulose-filter-binding assays for DNA interaction.
- Exonuclease and endonuclease activity assays.
Main Results:
- A purification protocol yielded Mu GAM from inclusion bodies.
- Mu GAM exhibited high affinity for linear double-stranded DNA.
- Mu GAM showed weaker binding to supercoiled and single-stranded DNA.
- Mu GAM effectively protected linear DNA against exonucleases.
- Mu GAM demonstrated limited inhibition of endonuclease activity.
Conclusions:
- Mu GAM can be efficiently purified using a guanidine hydrochloride denaturation-renaturation method combined with ion-exchange chromatography.
- Mu GAM preferentially binds to linear double-stranded DNA, particularly at its ends.
- Mu GAM confers protection against exonuclease degradation, supporting a model of end-binding activity.