Functional heterologous expression and purification of a mammalian methyl-CpG binding domain in suitable yield for DNA methylation profiling assays.
DNA methylation is a major epigenetic modification in mammalian cells, and patterns involving methylation of cytosine bases, known as CpG methylation, have been implicated in the development of many types of cancer. Methyl binding domains (MBDs) excised from larger mammalian methyl-CpG-binding proteins specifically recognize methyl-cytosine bases of CpG dinucleotides in duplex DNA. Previous molecular diagnostic studies involving MBDs have employed Escherichia coli for protein expression with either low soluble yields or the use of time-consuming denaturation-renaturation purification procedures to improve yields. Efficient MBD-based diagnostics require expression and purification methods that maximize protein yield and minimize time and resource expenditure. This study is a systematic optimization analysis of MBD expression using both SDS-PAGE and microscopy and it provides a comparison of protein yield from published procedures to that from the conditions found to be optimal in these experiments. Protein binding activity and specificity were verified using a DNA electrophoretic mobility shift assay, and final protein yield was improved from the starting conditions by a factor of 65 with a simple, single-step purification.
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
Mary E
ME
Boyd
Brandon W
BW
Heimer
Hadley D
HD
Sikes
D000818:Animals; D002846:Chromatography, Affinity; D004247:DNA / Q000737:chemistry; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D024202:Electrophoretic Mobility Shift Assay; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D051379:Mice; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D004268:DNA-Binding Proteins; C489323:Mbd1 protein, mouse; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D015254:DNA Modification Methylases