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Synthesis of Bioconjugates Using Sortase A as a Site-Specific Enzymatic Ligation Method.

Waller, Thomas. (2013) Synthesis of Bioconjugates Using Sortase A as a Site-Specific Enzymatic Ligation Method. Doctoral thesis, University of Surrey (United Kingdom)..

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The transpeptidase, Sortase A is an enzyme that has been used in this function previously ligate eGFP to polymer beads, short polymers and other proteins with great success. To further demonstrate the site specific and universal nature of Sortase A, the ligation reaction was expanded to a range of materials that included graft-to, graft-from and graft-through polymers and a number of short and self-assembling peptides. Enhanced green fluorescent protein (eGFP) and DNA-binding protein (Tus) were expressed with a LPTEGG tag for Sortase A mediated ligation followed by a Hexa-His tag, whilst Sortase A was expressed with a Hexa-His tag to aid with purification. The proteins, Sortase A and enhanced green fluorescent protein were expressed and purified successfully from Escherichia coli in yields of 141 mg in each case. The synthesis of a peptide initiator utilised two routes in the form of solution peptide synthesis and solid phase peptide synthesis. Both methods presented difficulties due to poor solubility of the peptide initiators and synthetic intermediates. The latter was more successful and involved the synthesis of a number of initiators containing diglycine and a bromo-isobutyrlamide group. The most successful of these was the peptide initiator GG[COCBr(CH3)2]-E-OH that was synthesised in a high yield (73%), was highly soluble in a number of solvents and was used effectively in ATRP and SET-LRP. Polymerisation involved a number of controlled radical polymerisation strategies including: ATRP, SET-LRP and RAFT. ATRP generated a number of peptide-polymers (98,723 g/mol, PDI 1.41, 20,387 g/mol, PDI 1.19 and 30,066 g/mol, PDI 1.45) that were suitable for attachment to the eGFP. SET-LRP also generated a number of peptide-polymers that were suitable for attachment to the eGFP in the graft-to (20,066 g/mol, PDI 1.26) and graft-through strategies (26,861 g/mol, PDI 1.19) and for use in peptide self-assembly (34,465 g/mol, PDI 1.52). RAFT was also used to test the possibility of post-polymerisation modification of the peptide-polymer (30,066 g/mol, PDI 1. 45) for use in conjugation to eGFP. Graft-to and through conjugation of the peptide-polymers from ATRP and SET-LRP to eGFP was successfully mediated by Sortase A. The grafting of pOEGMA from eGFP-peptide initiator using ATRP and SET-LRP also successfully generated protein-polymer conjugates. This demonstrates that Sortase A and the peptide initiator can be used as a universal method for graft-to, graft-from and graft-through conjugation. The grafting methodologies showed that conjugates synthesised using the graft-from had a higher yield during ligation compared to the graft-to conjugates. Site-specific conjugation of DNA-binding protein (Tus) to the self-assembling di-gly peptide had no effect on the morphology of the hydrogel and the DNA-binding protein Tus was still accessible within the gel network to complimentary binding sequences. With Sortase A it is possible to synthesise protein-polymer conjugates using a number of grafting methodologies. It is possible due to the universality of the method to transfer the technology to other proteins and polymers for efficient synthesis of novel protein-polymer conjugates.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Waller, Thomas.
Date : 2013
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 2013.
Depositing User : EPrints Services
Date Deposited : 14 May 2020 14:56
Last Modified : 14 May 2020 15:09

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