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
2004
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
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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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Author:
Karen-Anne Thomas
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