ABSTRACT
The Ig-binding properties of protein L from Peptostreptococcus magnus and protein G from Streptococcus have been successfully combined through the construction of a novel hybrid protein, consisting of a single Ig-binding domain from each protein. The biophysical and biochemical properties of this construct have been characterized through equilibrium and pre-equilibrium fluorescence spectroscopy, circular dichroism, isothermal titration calorimetry, affinity chromatography, and conformational stability studies using a chemical denaturant in order to examine the structure and availability of ligand binding sites in each domain. These studies show that despite the small size of the protein (Mw=16.5 kDa) each domain behaves in an independent manner with respect to the binding characteristics of the same domain in isolation.
Subject(s)
Bacterial Proteins/metabolism , Immunoglobulins/metabolism , Protein Engineering , Receptors, Immunologic/metabolism , Amino Acid Motifs , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Base Sequence , Binding Sites , Genetic Vectors , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin kappa-Chains/immunology , Immunoglobulin kappa-Chains/metabolism , Immunoglobulins/immunology , Molecular Sequence Data , Peptostreptococcus/immunology , Receptors, Immunologic/chemistry , Receptors, Immunologic/immunology , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Streptococcus/immunologyABSTRACT
A kappa-light chain from a Fab expression system was truncated by the insertion of a stop codon in the gene sequence to produce a variable light (VL) single domain antibody (dAb). Here, we describe the expression of dAb in the periplasm of Escherichia coli through fermentation in a defined media. Immunoglobulin binding domains from peptostreptococcal protein L (PpL) have been shown to bind specifically to kappa-light chains. We have produced recombinant PpL, at high yield, and this was used to custom-produce PpL-Sepharose affinity columns. Here, we show that the affinity purification of VL dAb by this method is simple and efficient with no apparent loss in protein at any stage. The truncated dAb protein product was confirmed by electrospray mass spectrometry and N-terminal sequencing. When analyzed by SDS-PAGE it was shown to be over 95% pure and produced at yields of 35-65 mg/L of culture medium. The dAb protein produced was shown by NMR and CD to be a folded beta-sheet domain. This domain is bound by PpL with a Kd of approximately 50 nM as determined by stopped-flow fluorimetry.
