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Applications of Sortase A from Staphylococcus aureus

Applications of Sortase A from Staphylococcus aureus
Applications of Sortase A from Staphylococcus aureus
Sortase A (SrtA) mediated ligation provides a mild and site-specific method for attaching a wide range of molecular probes to biological molecules, such as proteins and DNA. This method does not require any toxic reagents or harsh conditions to achieve high level of labelling, and the product could be easily isolated from the reaction mixture. This new labelling techniques could potentially improve exisiting technologies for studying molecular interations. A number of cloning and protein expression systems for the SrtA system were developed and the resulting proteins were very stable and in high purity.
The use of this technique for site-specific protein labelling was investigated. A range of molecules, such as fluorecein derivitives and 25 bp double-stranded DNA, were
successfully ligated to the target proteins in good yield. The use of the SrtA system in protein immobilisation was also thoroughly studied. A number of proteins with distintly
different functionalties, such as fluorescent proteins (BFP, EGFP, DsRed), enzyme (Fpr) and DNA-binding protein (Tus), were successfully immobilised onto highly cross-linked polymeric beads (GMA), soft-polymer resin matrix (Affi-gel) and glass surfaces. The amount of non-specifically bound protein onto these surfaces was found to be neglectable and the activity of the attached protein was retained.
Chan, Lok See
ad93b056-69b0-4ccd-9f6d-2ee539082243
Chan, Lok See
ad93b056-69b0-4ccd-9f6d-2ee539082243
Neylon, Cameron
697f067b-db25-4c41-9618-28f4b74f73aa

Chan, Lok See (2010) Applications of Sortase A from Staphylococcus aureus. University of Southampton, School of Chemistry, Doctoral Thesis, 188pp.

Record type: Thesis (Doctoral)

Abstract

Sortase A (SrtA) mediated ligation provides a mild and site-specific method for attaching a wide range of molecular probes to biological molecules, such as proteins and DNA. This method does not require any toxic reagents or harsh conditions to achieve high level of labelling, and the product could be easily isolated from the reaction mixture. This new labelling techniques could potentially improve exisiting technologies for studying molecular interations. A number of cloning and protein expression systems for the SrtA system were developed and the resulting proteins were very stable and in high purity.
The use of this technique for site-specific protein labelling was investigated. A range of molecules, such as fluorecein derivitives and 25 bp double-stranded DNA, were
successfully ligated to the target proteins in good yield. The use of the SrtA system in protein immobilisation was also thoroughly studied. A number of proteins with distintly
different functionalties, such as fluorescent proteins (BFP, EGFP, DsRed), enzyme (Fpr) and DNA-binding protein (Tus), were successfully immobilised onto highly cross-linked polymeric beads (GMA), soft-polymer resin matrix (Affi-gel) and glass surfaces. The amount of non-specifically bound protein onto these surfaces was found to be neglectable and the activity of the attached protein was retained.

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More information

Published date: 22 June 2010
Organisations: University of Southampton

Identifiers

Local EPrints ID: 173971
URI: https://eprints.soton.ac.uk/id/eprint/173971
PURE UUID: 4c8b3106-341a-410d-b34f-43f192702e82

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Date deposited: 19 May 2011 10:48
Last modified: 18 Jul 2017 12:12

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Contributors

Author: Lok See Chan
Thesis advisor: Cameron Neylon

University divisions

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