title: Importance of the linker in expression of single-chain Fv antibody fragments: optimisation of peptide sequence using phage display technology.
abstract: We have investigated the potential for enhancing the production of functional single-chain Fv antibody fragments (sFv), by altering the sequence of the linker that joins the variable domains of the molecule. To identify new functionally improved linkers we have used a phage display library containing a random sequence of six amino acids in the linker. Multiple rounds of panning on the antigen led to the selection of six different linker sequences that enhanced the binding of phage to the antigen. Five of the linkers also improved the secretion of soluble sFv by approximately five-fold. Analysis of the predominant linker sequence isolated showed that this improvement is not due to an increased affinity for the antigen, nor to alterations in the toxicity to bacteria. However the linker did affect the denaturation of the sFv in urea. It is therefore possible that the novel sequence helps in the refolding or secretion of the molecule.
authors.affiliation: Department of Immunology, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK.
authors.forename: D J
authors.initials: DJ
authors.lastname: Turner
authors.forename: M A
authors.initials: MA
authors.lastname: Ritter
authors.forename: A J
authors.initials: AJ
authors.lastname: George
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; C055966:immunoglobulin Fv