Experimental procedures forsynthesis, refolding and crystallization of Caspase-1.

Hoekstra, M.H. (2015) Experimental procedures forsynthesis, refolding and crystallization of Caspase-1. Master's Thesis / Essay, Biology.

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Abstract

Caspase-1, also known as IL-1-converting enzyme (ICE), is a key protein in the inflammatory process, and an interesting target to prevent undesired inflammation. Caspase-1 is a protease responsible for activating pro-inflammatory cytokine IL1ß by cleaving pro-IL1ß into active inflammatory molecule IL1ß. In addition, Caspase-1 causes rapid cell death in macrophages that contain intracellular bacteria, which induces response against bacterial infections. In summary, Caspase-1 is a key inflammatory mediator for the host response to infection, injury and disease. Though the inflammatory response is vital to the host, IL1ß-driven inflammation often has a disastrous effect during disease and/or brain injury, conditions that have limited clinical options. This makes Caspase-1 an interesting therapeutic target to mitigate autoimmune response) 1. Previously discovered inhibitors of Casapase-1 bind covalently to the protein. This covalent nature of binding resulted in toxicity and did not pass clinical trials) 2. To make the protein suitable for testing against possible leads to inhibit its function, Caspase-1 can be produced through protein expression, refolding and crystallization. Because conditions based on public protocols were hard to establish the goal of this project was to find a method to express Caspase-1 from its gene, refold it into its proper conformation and co-crystallize with a number of possible lead compounds (ALC150, ALC129, VX765 and AD4). Compounds used for co-crystallization have been produced by A. Chandgude in the drug design lab of Groningen University. In this project Caspase-1 was expressed, purified and folded into its natural shape. Correct folding was demonstrated by circular dichroism. Caspase-1 was crystallized with & without compounds, diffraction data has been collected and structure solutions are underway.

Item Type:	Thesis (Master's Thesis / Essay)
Degree programme:	Biology
Thesis type:	Master's Thesis / Essay
Language:	English
Date Deposited:	15 Feb 2018 08:09
Last Modified:	15 Feb 2018 08:09
URI:	https://fse.studenttheses.ub.rug.nl/id/eprint/13413

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