Overexpression and affinity chromatography purification of the Type III restriction endonuclease EcoP15I for use in transcriptome analysis.
The Type III restriction endonuclease EcoP15I is a multifunctional hetero-oligomeric enzyme that recognizes the non-symmetric DNA sequence 5'-CAGCAG. For efficient cleavage, EcoP15I needs the interaction with two copies of the recognition sequence that have to be inversely oriented in the DNA double strand. The enzyme cuts the upper DNA strand 25-26 bp and the lower DNA strand 27-28 bp, respectively, downstream of the recognition sequence-a distinct feature that makes the enzyme particularly valuable for gene expression profiling methods relying on the SAGE procedure (Matsumura et al., PNAS 100, 15718, 2003). Because the broader use of this transcriptome analysis method requires the availability of larger amounts of restriction endonuclease EcoP15I and the enzyme is not commercially available, we have cloned the genes coding for the EcoP15I restriction endonuclease into pQE-16 plasmid vector that provides the enzyme with a C-terminal 6xHis-tag. After Ni-NTA affinity chromatography and ion exchange chromatography on heparin sepharose, we obtained 5mg homogeneous EcoP15I per gram cell pellet within 1-2 day(s). Moreover, the C-terminally 6xHis-tagged EcoP15I restriction endonuclease shows comparable enzymatic activity as the untagged enzyme.
Möncke-Buchner
Elisabeth
E
Institute of Virology, Humboldt University Medical School (Charité), 10098 Berlin, Germany.
Mackeldanz
Petra
P
Krüger
Detlev H
DH
Reuter
Monika
M
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D020543:Proteome / Q000032:analysis*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014157:Transcription Factors / Q000032:analysis*
D020543:Proteome; D011994:Recombinant Proteins; D014157:Transcription Factors; C089712:DNA modification methylase EcoP15I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes