Expression and Purification of Wild Type and Mutant bZIP Transcription Factors to Analyze DNA Binding Specificity
Faculty Mentor Name
Jerry Tsai- faculty mentor, Maggie Klemer De Lasse- Graduate mentor
Research or Creativity Area
Natural Sciences
Abstract
Gene expression regulation depends on transcription factors that recognize specific DNA sequences. However, the mechanism by which these proteins achieve sequence-specific recognition is not fully understood. Basic leucine zipper (bZIP) transcription factors, including CREB and C/EBPa, are simple α-helical proteins characterized by a leucine zipper dimerization domain and a basic DNA binding region that interacts directly with specific DNA bases. Using the Knob Socket (KS) analysis, a quadrapartite model was developed characterizing the amino acid specific hydrophobic packing that recognizes individual DNA bases. To experimentally test this model, both wild-type and mutant proteins were expressed and purified. Plasmids, encoding wild-type and mutant bZIP proteins, contained an antibiotic resistance selectable marker, a T7 promoter regulated by the lac operator, and a 6xHIS-SUMO purification system. These elements allow for selection and induction of bacterial cells only containing our plasmid of interest as well as protein purification. Plasmids were generated via Gibson Reaction and transformed into DH5a Escherichia coli (E.coli) cells for cloning. Miniprep plasmid purification preceded transformation into BL21 E.coli for protein expression. Transformed colonies were grown overnight and subcultured. Protein expression was induced using IPTG, an allolactose mimic, at mid-log phase (OD600 ~0.6-0.8). Cells were lysed via sonication, and the soluble protein lysate was collected. His-tagged proteins were purified by nickel affinity chromatography and eluted with increasing imidazole concentrations. Elution fractions were analyzed by Polyacrylamide Gel Electrophoresis (SDS-PAGE) to assess protein size and purity. Distinct bands at the expected molecular weight confirmed successful expression and purification of both wild-type and mutant proteins, with ongoing efforts to improve protein purity. These purified proteins will be used in Electrophoretic Mobility Shift Assays (EMSA) to analyze the influence of mutations on bZIP-DNA binding specificity and evaluate how well the KS model explains protein-DNA recognition.
Purpose
Protein-DNA specificity is incredibly important in the scope of understanding diseases, especially in the context of transcription factors, though very little is understood about how recognition occurs. In order to identify and understand the "code" to protein-DNA binding, experimental techniques were performed to complement previous computational work.
Expression and Purification of Wild Type and Mutant bZIP Transcription Factors to Analyze DNA Binding Specificity
Gene expression regulation depends on transcription factors that recognize specific DNA sequences. However, the mechanism by which these proteins achieve sequence-specific recognition is not fully understood. Basic leucine zipper (bZIP) transcription factors, including CREB and C/EBPa, are simple α-helical proteins characterized by a leucine zipper dimerization domain and a basic DNA binding region that interacts directly with specific DNA bases. Using the Knob Socket (KS) analysis, a quadrapartite model was developed characterizing the amino acid specific hydrophobic packing that recognizes individual DNA bases. To experimentally test this model, both wild-type and mutant proteins were expressed and purified. Plasmids, encoding wild-type and mutant bZIP proteins, contained an antibiotic resistance selectable marker, a T7 promoter regulated by the lac operator, and a 6xHIS-SUMO purification system. These elements allow for selection and induction of bacterial cells only containing our plasmid of interest as well as protein purification. Plasmids were generated via Gibson Reaction and transformed into DH5a Escherichia coli (E.coli) cells for cloning. Miniprep plasmid purification preceded transformation into BL21 E.coli for protein expression. Transformed colonies were grown overnight and subcultured. Protein expression was induced using IPTG, an allolactose mimic, at mid-log phase (OD600 ~0.6-0.8). Cells were lysed via sonication, and the soluble protein lysate was collected. His-tagged proteins were purified by nickel affinity chromatography and eluted with increasing imidazole concentrations. Elution fractions were analyzed by Polyacrylamide Gel Electrophoresis (SDS-PAGE) to assess protein size and purity. Distinct bands at the expected molecular weight confirmed successful expression and purification of both wild-type and mutant proteins, with ongoing efforts to improve protein purity. These purified proteins will be used in Electrophoretic Mobility Shift Assays (EMSA) to analyze the influence of mutations on bZIP-DNA binding specificity and evaluate how well the KS model explains protein-DNA recognition.
