High-throughput screening technologies for identification and expression of functional domains of proteins of biomedical importance

Parry-Morris, Sarah (2015) High-throughput screening technologies for identification and expression of functional domains of proteins of biomedical importance. Doctoral thesis (PhD), University of Sussex.

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Abstract

The ability to produce multi-milligram quantities of a recombinant ‘target’ protein in a proteolytically stable, soluble, and functional form is often necessary for subsequent biochemical, biophysical and structure-based analyses. Sub-constructs expressing only part of a large target protein can often be useful. Combinatorial Domain Hunting (CDH) is a methodology that allows the rapid production of sub- constructs via a random DNA fragmentation technique. One particular issue with CDH is that it can be used to identify globular regions or domains of a target protein, but does not take account of the functional properties of such domains; therefore some ‘hits’ are not useful, because they exclude these functional regions. Here, we have attempted to enhance the CDH methodology by including an additional screening step that could specifically identify those constructs expressing functional protein domains. However, whilst rigorous testing of this functionality screen proved it to be successful under selective conditions, it was not considered suitable for inclusion in the CDH method.

CDH was also used to identify highly expressed, proteolytically stable regions of a previously largely uncharacterized protein, and to investigate their functionality. Human Claspin is a large, highly charged, S=phase specific ‘molecular scaffold’ protein, with no identifiable sub-domains or enzymatic function(s). However, Claspin is known to make multiple different protein-protein interactions at replication forks during the intertwined processes of DNA replication and DNA replication-coupled repair. CDH successfully identified a number of N-terminal expression constructs that could be expressed and purified to a high degree of homogeneity. Structural and functional analyses of these protein fragments indicated that the N-terminus of human Claspin is intrinsically disordered, and elongated in nature. However, these regions may become ordered upon binding to their respective protein or macromolecular partner(s). Furthermore, several N-terminal fragments were found to be able to bind to both single- or double-stranded DNA when longer than 16 nucleotides/base-pairs in length. Additionally, the phospho-specific protein-protein interaction made by human Claspin, with the checkpoint kinase Chk1 was further investigated.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Depositing User: Library Cataloguing
Date Deposited: 23 Dec 2015 10:27
Last Modified: 23 Dec 2015 10:27
URI: http://sro.sussex.ac.uk/id/eprint/58908

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