Event Information
DISCOVERY OF INSULIN CAPTURE BY GENOME-INSPIRED DNA IN AFFINITY MALDI AND AFFINITY CHROMATOGRAPHY
- Abstract:
- Several proteins have been shown to interact with G-quartet DNA in vitro and G-quartet formation has been proposed to play multiple roles in human cells in vivo. To this end, the non-coding Insulin Linked Polymorphic Region (ILPR), which is just upstream of the human insulin gene, contains a tandem repeat of a guanosine rich sequence that has been shown to form a G-quartet structure in vitro. It has been proposed that G-quartet structure in this region is involved in the replication slippage that is associated with insulin-dependant diabetes mellitus, as well as impacting the transcriptional levels of insulin.
The aim of this project was to develop a directed proteomic approach for identifying proteins that may interact with G-quartet structures in genomic DNA. Thrombin and its G-quartet forming oligonucleotide ligand serve as a model system for developing a methodology to screen for G-quartet-binding proteins in cell lysates. Preliminary work includes introduction of thrombin solutions into G-quartet coated capillaries for affinity chromatography. Thrombin was shown to be captured, washed, and eluted under conditions that unfold the G-quartet DNA. The eluate was collected and analyzed to determine the protein content and identity using fluorescence spectroscopy and mass spectrometry, respectively.
The affinity chromatography methodology was then utilized to discover proteins that interact with the genome-inspired ILPR sequence. A second affinity screening method, affinity MALDI, was also utilized to search for ILPR-binding proteins. The results of both methodologies showed that human insulin was captured from an ILPR-modified surface in both purified solution as well as in the human pancreas nuclear extracts. Isothermal titration calorimetry and fluorescence spectroscopy were used to estimate a binding constant for the insulin-ILPR complex corresponding to 1 and 2 μM, respectively.
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