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The Ohio State University

1. Sarkar, Mohosin M. Engineering Proteins with GFP: Study of Protein-Protein Interactions In vivo, Protein Expression and Solubility.

Degree: PhD, Chemistry, 2009, The Ohio State University

Protein–protein interactions (PPIs) play a key role in most biological processes. Many of these interactions are necessary for cell survival. To understand the molecular mechanisms of biological processes, it is essential to study and characterize protein-protein interactions, identify interacting partners and protein interaction networks. There are a number of methods that have been developed to study protein-protein interactions in vitro and in vivo, such as yeast-2-hybrid, fluorescence resonance energy transfer, co-immunoprecipitation, etc. Split protein reassembly is an in vivo probe of protein interactions that circumvents some of the problems with yeast 2-hybrid (indirect interactions, false positives) and co-immunoprecipitation (loss of weak and transient interactions, decompartmentalization). Split GFP reassembly is especially attractive because the GFP chromophore forms spontaneously on protein folding in almost every cell type. However, existing split systems have limitations of evolving cellular fluorescence slowly (3-4 days), failure to evolve at all for some interactions, and also failure to work at a physiological temperature. Among different variants of GFP tested, we found that split folding-reporter GFP (frGFP, a hybrid of EGFP and GFPuv) evolves fluorescence much faster (24 - 30 h) with associating peptides and also evolves fluorescence for the RING domain BRCA1/BARD1 wild type pair. Thirty six known cancer-associated BRCA1 RING domain mutants were tested with split-frGFP system for their role in BRCA1/BARD1 interactions. Some of these mutations resulted in significant reduction of complex reassembly and cellular fluorescence. Split frGFP fragments were further improved by directed evolution (error-prone PCR and DNA shuffling) to obtain fragments for fast and efficient fluorescence reassembly. The evolved fragments were able to generate fluorescence in as little as 12-16 h at 30 °C and in 10-14 h at 37 °C. This system was successfully tested for the detection of interactions of several therapeutically important protein pairs (such as Bcl-xL/Bim, Bcl-2/Bim, p53/hDM2, XIAP/Smac), which have key roles in apoptosis and cancer. Response to known inhibitors of these interactions was also tested using this system. These results suggest that the efficient split GFP (esGFP) fragments we developed will be very useful for in vivo screening of small molecule or cyclic peptide libraries to develop effective modulators of protein-protein interactions in their native cellular context from direct fluorescence reassembly. Human paraoxonase-1 (huPON1) has been known for some time for its broad hydrolytic specificity against organophosphorus (OP) pesticides and nerve agents, such as, sarin, soman and tabun, etc. The large-scale expression of the soluble protein and the improvement of the stability and catalytic activity are the most critical challenges for huPON1 to be used as a drug for detoxification of OP pesticides and nerve agents. As a human protein, it is considered to be a potent candidate for… Advisors/Committee Members: Magliery, Thomas (Advisor).

Subjects/Keywords: Biochemistry; Chemistry; Molecular Biology; Protein-protein interactions; Split GFP assay; Split GFP reassembly; BRCA1/BARD1 interactions; BRCA1 cancer associated mutations; Human paraoxonase-1

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APA (6th Edition):

Sarkar, M. M. (2009). Engineering Proteins with GFP: Study of Protein-Protein Interactions In vivo, Protein Expression and Solubility. (Doctoral Dissertation). The Ohio State University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=osu1261418776

Chicago Manual of Style (16th Edition):

Sarkar, Mohosin M. “Engineering Proteins with GFP: Study of Protein-Protein Interactions In vivo, Protein Expression and Solubility.” 2009. Doctoral Dissertation, The Ohio State University. Accessed August 14, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1261418776.

MLA Handbook (7th Edition):

Sarkar, Mohosin M. “Engineering Proteins with GFP: Study of Protein-Protein Interactions In vivo, Protein Expression and Solubility.” 2009. Web. 14 Aug 2020.

Vancouver:

Sarkar MM. Engineering Proteins with GFP: Study of Protein-Protein Interactions In vivo, Protein Expression and Solubility. [Internet] [Doctoral dissertation]. The Ohio State University; 2009. [cited 2020 Aug 14]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=osu1261418776.

Council of Science Editors:

Sarkar MM. Engineering Proteins with GFP: Study of Protein-Protein Interactions In vivo, Protein Expression and Solubility. [Doctoral Dissertation]. The Ohio State University; 2009. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=osu1261418776

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