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Cloning and recombinant protein expression of the human Prmt5 gene

for the understanding of the histone arginine methyltransferase pathway of PRMT5

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Cloning and recombinant protein expression of the human Prmt5 gene

for the understanding of the histone arginine methyltransferase pathway of PRMT5

Rechten: Alle rechten voorbehouden

Samenvatting

The human protein arginine methyltransferases (PRMTs) family consists of 11 members. All of the PRMT members modify the characteristics of cellular proteins by transferring a methyl-group to the side chain of arginine amino acid in target proteins, which are important for both the normal and malign cellular processes. An increasing number of studies link PRMT5 no numerous pathologies that include cancers. Moreover, little is known about the PRMT5 pathway. Therefore, we focus on understanding the structure, the exact cellular functions and the regulation of PRMT5.
PRMT5 is an arginine methyltransferase enzyme that methylates arginine on histones H3 and H4 at the loci R8 and R3 respectively, by transferring one or two methyl groups to the arginine side chains. Histones are the stage of diverse post-translational modifications that ultimately regulates the gene transcription. Arginine methylation is one prominent feature of the post-translational histone modifications in the regulation of chromatin structure and function. Arginine methylation, or any of the other histone modifications, can have both activating and repressive functions on the transcription, which can lead in changing the chromatin status to an unregulated chromatin. Chromatins are a combination of DNA, histones and proteins. Transcription coregulators (TCs), bind to a nuclear receptor and play a key role in regulating of the compaction levels of the DNA in the chromatin. Tight packing will inhibit the transcription events. At the opposite, looser packing will permit the binding of the large complex of proteins which form the transcription factor and activate the gene expression.
PRMT5 is key in the regulation of the circadian cycle by modulating the regulation of expression of clock-genes. Recently, a trail of evidence link deregulated PRMT5 to the onset of carcinogenetic events. However, little is known about the exact biological functions and regulations of the PRMT5, as well as the PRMT family.
My research in the Di Luccio research group in South Korea was centered on understanding the structure of PRMT5. In this report, I describe the cloning of human Prmt5 and recombinant protein expression of a pure PRMT5 protein for functional and structural studies. The expected results will greatly add to our understanding of the human histone arginine methyltransferase pathways in general. My project is the first step of a multi-year team effort aiming at understanding the histone code, to unravel the regulatory cross-talks events within transcription co-regulators and the design of selective and specific drugs designed to modulate the activity of PRMT enzymes.
The primarily objective of this multi-year project is to solve the unknown protein structure of the human PRMT5 in order to better understand its function, mechanism and regulation. The first goal of my project was achieved by successfully cloning the human Prmt5 gene and by building a recombinant expression vector for recombinant protein expression in E.coli. Next, I determined the optimum parameters for recombinant protein expression in E.coli BL21 strain. I successfully over-expressed human PRMT5 and purified it by affinity chromatography. I performed peptide mass fingerprinting by MALDI-TOF mass spectrometry to confirm PRMT5 on SDS gels. After introducing an ATP treatment to wash off HSP70, an E.coli chaperone protein, the PRMT5 showed no contamination anymore. With the acquired clean protein crystallization trays were set up. In one condition crystals were big enough for harvesting, but too small to determine the protein structure by X-ray crystallography. This technique relies on growing protein crystals able to diffract at atomic resolution under a stream of X-ray radiations. The prerequisite for the growth of protein crystals is a pure protein sample.

Toon meer
OrganisatieAvans Hogeschool
AfdelingATGM Academie voor de technologie van Gezondheid en Milieu
PartnersKyungpook National University, Daegu, South Korea; School of Applied Biosciences
Datum2011-07
TypeBachelor
TaalEngels

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