Recombinant production of a VL single domain antibody in Escherichia coli and analysis of its interaction with peptostreptococcal protein L.
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.
Cossins
Aimee J
AJ
School of Biological Sciences, University of Southampton, Bassett Crescent East, SO16 7PX, UK.
Harrison
Steven
S
Popplewell
Andrew G
AG
Gore
Michael G
MG
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000276:immunology; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D005470:Fluorometry; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000096:biosynthesis* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000276:immunology* / Q000302:isolation & purification; D017434:Protein Structure, Tertiary
D000906:Antibodies; D001426:Bacterial Proteins; C055047:Ig L-binding protein, Peptostreptococcus; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D010446:Peptide Fragments