A rapid purification procedure for the HsdM protein of EcoR124I and biophysical characterization of the purified protein

► A rapid purification method, producing 20mg of protein from 5g cell paste. ► A novel application of desalting columns to achieve a high degree of purity. ► The method may be of general relevance for purification of DNA-binding proteins. ► The HsdM protein exists in a concentration dependent monome...

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Bibliographic Details
Published in:Protein expression and purification 2013-02, Vol.87 (2), p.136-140
Main Authors: Taylor, James E.N., Swiderska, Anna, Geoff Kneale, G.
Format: Article
Language:English
Subjects:
Analytical ultracentrifugation
Bacterial Proteins - biosynthesis
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - isolation & purification
Deoxyribonucleases, Type I Site-Specific - chemistry
DNA methyltransferases
DNA Restriction-Modification Enzymes - biosynthesis
DNA Restriction-Modification Enzymes - chemistry
DNA Restriction-Modification Enzymes - genetics
DNA Restriction-Modification Enzymes - isolation & purification
Escherichia coli
Methyltransferases - biosynthesis
Methyltransferases - chemistry
Methyltransferases - genetics
Methyltransferases - isolation & purification
Protamines - chemistry
Protein Subunits
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Sodium Chloride - chemistry
Type I restriction enzymes
Ultracentrifugation
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Summary:► A rapid purification method, producing 20mg of protein from 5g cell paste. ► A novel application of desalting columns to achieve a high degree of purity. ► The method may be of general relevance for purification of DNA-binding proteins. ► The HsdM protein exists in a concentration dependent monomer-dimer equilibrium. Type I restriction–modification (R–M) systems are comprised of two multi-subunit enzymes with complementary functions: the methyltransferase (∼160kDa), responsible for methylation of DNA, and the restriction endonuclease (∼400kDa), responsible for DNA cleavage. Both enzymes share a number of subunits, including HsdM. Characterisation of either enzyme first requires the expression and purification of its constituent subunits, before reconstitution of the multisubunit complex. Previously, purification of the HsdM protein had proved problematic, due to the length of time required for the purification and its susceptibility to degradation. A new protocol was therefore developed to decrease the length of time required to purify the HsdM protein and thus prevent degradation. Finally, we show that the HsdM subunit exhibits a concentration dependent monomer–dimer equilibrium.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2012.11.003