Mineral binding peptides by phage display: experimental and bioinformatics studies

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Thota, V., 2019. Mineral binding peptides by phage display: experimental and bioinformatics studies. PhD, Nottingham Trent University.

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Abstract

Phage display has attracted a great deal of interest in the identification of peptides specific to nanomaterials revealing distinctive binding behaviour. Though significant progress has been made in selecting and screening of biomolecule binding peptides, the accuracy of molecular recognition for inorganic materials is still challenging due to the limitations of phage display libraries and biopanning process. The study presented in this thesis is aimed at isolating mineral binding peptides by phage display and verifying them experimentally and/ or bioinformatically; exploring the role of electrostatic/ non-electrostatic interactions in the aqueous phase and the factors responsible for the adsorption or desorption of peptide or phage from the mineral surface.

Firstly, silica binding peptides LPVRLDW, NDLMNRA, GQSEKHL and GASESYL have been identified using the phage display technique by varying experimental conditions including pH, detergent, washing and elution buffers to remove unique 7-mer peptide binding phages from amorphous hydrophilic silica nanoparticles via disruption of the molecular interactions between the phage attached peptides and the nanoparticles. A repanning method reported here, has experimentally reproduced the majority of the initially discovered silica binders; alongside identifying/ recovering additional peptide sequences HYIDFRW, KIAVIST and YSLKQYQ that may have been overlooked in the routine approach to biopanning. Secondly, an alternative three step elution method reported here, has eluted and recovered most target silica binders including ADIRHIK in the early panning rounds and removed the phage clones that are bound to silica by hydrophobic, hydrogen bonding and electrostatic attractions or repulsions; as opposed to one specific buffer being used for all panning rounds including elution steps in traditional biopanning experiments. Also, the phage clones that resist detection to single elution step have been eluted in the other successive elution steps, thereby recovering and improving the elution procedure for silica surfaces. In addition, these three different elution buffers have eluted phage clones that are interaction or charge specific subject to change in the elution buffer pH condition. The experimental results demonstrate that this sequential three step elution process was able to isolate tightly bound target silica binders in one or two biopanning rounds than the more typical four to five; thereby reducing biopanning rounds, cost and effort. Moreover, the bioinformatic analysis to cross check the authenticity/ quality of target binders has been reported.

Furthermore, selected silica binding peptides isolated from phage display experiments were synthesized by a solid phase peptide synthesis approach and peptide-silica interactions explored in vitro, using quantitative and qualitative techniques. The fluorometric analysis of these peptides revealed that the peptide adsorption to silica surfaces would have more than one type of interactions (i.e. electrostatic/ hydrophobic/ H-bonding and Van der Waals) and could be influenced by the experimental conditions. More significantly, an increase in binding activity to negatively charged silica nanoparticles was noticed for the peptides (HYIDFRW, KIAVIST and YSLKQYQ) modified with an amide (NH2) group as opposed to a carboxyl group at the C-terminal end; driving an increase in overall charge or pI of the peptides. Insights from the studies presented may provide valuable information for designing and engineering of silica directed constructs for a range of biomedical and nanotechnological applications.

Item Type:	Thesis
Creators:	Thota, V.
Date:	January 2019
Rights:	This work is the intellectual property of the author. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed to the owner of the Intellectual Property Rights.
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	01 May 2019 12:50
Last Modified:	01 May 2019 12:50
URI:	https://irep.ntu.ac.uk/id/eprint/36373