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HALAWA, MOHAMED and UNSPECIFIED (2020) Novel fusion tags for protein production, purification and research applications. PhD thesis, University of Nottingham.

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Abstract

Soluble production of recombinant proteins is required for the study of structure and function of these proteins as well as for future therapeutic applications. Unfortunately, many proteins have a propensity to be expressed as insoluble proteins when expressed in a heterologous host, including Escherichia coli. The use of solubility tags is one of the strategies to overcome this difficulty.

The first aim of this thesis was to design and investigate new candidate fusion tags for protein expression in E. coli and as potential carrier mediated protein crystallization aids. Two fusion tag protein candidates had previously been identified from the Protein Data Bank (PDB) and literature searches based on criteria such as shape, size, ease of expression and crystallisation and the location of termini. These new fusion tags were the receiver domain of Myxococcus xanthus social motility protein FrzS (PDB code 2GKG) and the calponin homology domain from human β-spectrin (PDB code 1BKR). These tags proteins were fused to chosen human model proteins and peptides to assess expression, solubility and purification. Three groups of model proteins were selected for investigation: two weakly expressed proteins of unknown structuresthat display solubility issues (human Bardet Biedl protein 5, ubiquitin specific protease 20 zinc finger domain), two highly expressed proteins of known structures (ubiquitin carboxyl-terminal hydrolase isozyme L1, ubiquitin carboxyl-terminal hydrolase isozyme L3) and two peptides which are difficult to express without solubility tags, as the peptides have a high susceptibility for degradation. These two peptides (Ataxin residues 278-29 and α-synuclein residues 58-79) have been successfully obtained using Maltose binding protein (MBP) fusions. These proteins were expressed ii as soluble proteins and were more amenable for purification when 2GKG and 1BKR tags were inserted at the fusion partners amino-terminal end. The results obtained in terms of solubility and purification with these novel tags compared favourably with results obtained using the widely used solubility tag Maltose binding protein (MBP) tag. Thus, these our two novel fusion tags have the ability to produce soluble proteins almost near to that level obtained by using MBP tag when they fused with these chosen model proteins and peptides. The fusion proteins produced were also investigated for protein crystallization, but no crystals have been obtained till now.

The second aim of this thesis was to design and investigate a new fusion tag utilizing the ubiquitin protein due to its high stability and solubility. Ubiquitin was engineered by insertion of a histidine tag into the loop between strands 3 and 4 to be used as affinity and solubility tag. The ubiquitin modified with a histidine tag inserted between the strands 3 and 4 (UBHIS3S4) was expressed and purified alone in a high yield but when fused to two model proteins BBS5(1-270) and USP20 ZNFUBP (1-108) showed relatively a low yield. These results suggested that UBHIS3S4 protein was not favourable for use as affinity and solubility tag in these two instances.

In addition, the ubiquitin protein was designed and evaluated as protein scaffold due to its high stability and solubility. A peptide sequence from a constrained peptide library had previously been identified by next generation phage display technology that interacts with the N-terminal domain of the ubiquitin specific protease 11 (USP11) residues (1-244). This peptide sequence was inserted between strands 3 and 4 of ubiquitin to assess the suitability of ubiquitin as a scaffold for peptide display. The modified ubiquitin was expressed and purified and binding studies with USP11 (1-244) iii were performed by isothermal titration calorimetry which confirmed the binding. The results suggested that ubiquitin is suitable as a scaffold to display peptides which are at least 9 amino acids length between strands 3 and 4. Use of ubiquitin as a protein scaffold could serve as the basis for the production of peptidomimetic drugs and for different studies on drug discovery as anticancer drugs.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Dreveny, Ingrid Emsley, Jonas
Keywords:	Fusion tag protein candidates, Ubiquitin, Protein Data Bank
Subjects:	Q Science > QD Chemistry > QD901 Crystallography Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools:	UK Campuses > Faculty of Science > School of Pharmacy
Item ID:	60559
Depositing User:	Halawa, Mohamed
Date Deposited:	31 Jul 2020 04:40
Last Modified:	31 Jan 2023 08:19
URI:	https://eprints.nottingham.ac.uk/id/eprint/60559

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