Construction and characterization of protein rLG, a novel 16.5 kDa hybrid protein with a large binding repertoire for immunoglobulin fragments
Harrison, S.L., Housden, N.G. and Gore, M.G. (2005) Construction and characterization of protein rLG, a novel 16.5 kDa hybrid protein with a large binding repertoire for immunoglobulin fragments. FEBS Journal, 272, (s1), p.254.
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Several proteins isolated from the surface of Gram-positive pathogenic bacteria have been shown to bind immunoglobulin (Ig) in a non-antigenic manner. The most widely studied of these proteins are protein A from Staphylococcus aureus, protein G from groups C and G streptococci, and protein L from Peptostreptococcus magnus. Although very useful reagents these all have limitations to their general applicability.Attempts have previously been made to broaden the binding spectra of individual Ig-binding molecules through the production of multidomain fusion proteins. The binding repertoires of proteins A and G restricts binding of a construct to specific Ig isotypes only. The ability of protein L to uniquely bind the variable domain of k-chains makes this protein a potential tool for the purification of Ig regardless of isotype. In the present study, a single k-chain binding domain of protein L was linked to a single Fc-binding domain of protein G to yield the novel, recombinant protein rLG gene. The rLG gene was cloned into the expression vector pKK223-3 allowing it to be over-expressed in E.coli JM103 cells. These studies show that despite its small size (Mr = 16.5 kDa), protein rLG exhibits simultaneous binding of ligands to both moieties, thereby making it a more versatile tool for Ig purification. A program of site directed mutagenesis (SDM) has been employed to characterize the binding properties and structural stability of protein rLG by equilibrium and stopped-flow fluorimetry, isothermal titration calorimetry, immuno-diffusion assay, affinity chromatography, circular dichroism and chemical denaturation studies.
|Subjects:||Q Science > QH Natural history > QH301 Biology|
|Divisions:||University Structure - Pre August 2011 > School of Biological Sciences
|Date Deposited:||06 Aug 2008|
|Last Modified:||01 Jun 2011 09:34|
|Contributors:||Harrison, S.L. (Author)
Housden, N.G. (Author)
Gore, M.G. (Author)
|Date:||1 July 2005|
|RDF:||RDF+N-Triples, RDF+N3, RDF+XML, Browse.|
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