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Characterisation of a bivalent Ig-binding domain from Peptostreptococcal protein L

Characterisation of a bivalent Ig-binding domain from Peptostreptococcal protein L
Characterisation of a bivalent Ig-binding domain from Peptostreptococcal protein L

Protein L is an elongated, fibrous protein of 76 - 106 kDa that is expressed on the cell surface of approximately 10% of Peptostreptococcus magnus strains.  It contains between 3 and 5 Ig-binding domains, which bind specifically and with high affinity to the VL portion of an Ig molecule.  The DNA of a single Ig-binding domain in protein L from strain 3316 has previously been cloned into a pKK22303 based vector and the protein product (PpL3316) has been expressed and purified from E. coli.  X-ray crystallography studies have shown that this protein contains two VL binding sites and subsequent kinetic experimentation has revealed that these interfaces have different binding affinities for κ-chain.  Stopped-flow fluorescence spectroscopy and isothermal titration calorimetry (ITC) have therefore been conducted to determine the relative affinity of each site in the presence of two different human κ-chain proteins and these studies have indicated that not only is the affinity of site 2 lower than that of site 1 but that the difference in relative affinity between the two sites varies with κ-chain.  Stopped-flow experimentation has suggested that there is a 54-fold difference in affinity between the sites with one κ1-chain (κTI) but only a 6-fold difference with another κ1-chain (κTX).  As a result of these findings a single Ig-binding domain in protein L from strain 312 (the B4 domain) has been isolated, using PCR, and the DNA has been cloned into the pKK223-2 vector.  The protein product (PpL312) has been expressed and purified from E. coli and stopped-flow fluorescence spectroscopy and ITC have been conducted.

University of Southampton
Thomas, Karen-Anne
Thomas, Karen-Anne

Thomas, Karen-Anne (2004) Characterisation of a bivalent Ig-binding domain from Peptostreptococcal protein L. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Protein L is an elongated, fibrous protein of 76 - 106 kDa that is expressed on the cell surface of approximately 10% of Peptostreptococcus magnus strains.  It contains between 3 and 5 Ig-binding domains, which bind specifically and with high affinity to the VL portion of an Ig molecule.  The DNA of a single Ig-binding domain in protein L from strain 3316 has previously been cloned into a pKK22303 based vector and the protein product (PpL3316) has been expressed and purified from E. coli.  X-ray crystallography studies have shown that this protein contains two VL binding sites and subsequent kinetic experimentation has revealed that these interfaces have different binding affinities for κ-chain.  Stopped-flow fluorescence spectroscopy and isothermal titration calorimetry (ITC) have therefore been conducted to determine the relative affinity of each site in the presence of two different human κ-chain proteins and these studies have indicated that not only is the affinity of site 2 lower than that of site 1 but that the difference in relative affinity between the two sites varies with κ-chain.  Stopped-flow experimentation has suggested that there is a 54-fold difference in affinity between the sites with one κ1-chain (κTI) but only a 6-fold difference with another κ1-chain (κTX).  As a result of these findings a single Ig-binding domain in protein L from strain 312 (the B4 domain) has been isolated, using PCR, and the DNA has been cloned into the pKK223-2 vector.  The protein product (PpL312) has been expressed and purified from E. coli and stopped-flow fluorescence spectroscopy and ITC have been conducted.

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Published date: 2004

Identifiers

Local EPrints ID: 465419
URI: http://eprints.soton.ac.uk/id/eprint/465419
PURE UUID: 8aa481f0-46b7-4ae6-a653-92dfb5406028

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Date deposited: 05 Jul 2022 00:51
Last modified: 05 Jul 2022 00:51

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Contributors

Author: Karen-Anne Thomas

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