title: Variable region sequence modulates periplasmic export of a single-chain Fv antibody fragment in Escherichia coli.
abstract: Using PCR, we have cloned antibody heavy and light chain variable region (VH and VL) coding sequences specific for a recombinant hepatitis B virus surface antigen (HBsAg) and assembled these for expression as single-chain Fv (scFv) fragments in Escherichia coli periplasm using the ompA signal peptide. The vectors also encoded N- or C-terminal His6 extensions to allow for the purification of the expressed proteins using immobilized metal affinity chromatography (IMAC). We found that the VH-linker-VL configuration of the scFv was not exported to the periplasm but remained associated with cellular insoluble material, from which it could be extracted, renatured to its active form by gentle dialysis and purified using IMAC. The molecular size of the scFv suggests that the ompA signal peptide was not processed. Based on previous reports, we hypothesized that the arginine in framework 1 (FR1) of the VH might interfere with translocation to the periplasm by means of the signal peptide. Because no arginines are present in FR1 of VL, we reversed the order of the V-regions in the scFv and observed efficient export of the active scFv to the periplasm. Furthermore, when the arginine in FR1 of VH was mutated to glycine in the original VH-linker-VL construct, active scFv was also exported to the periplasm. Thus, exposed positive charges near the signal peptide may account for at least some of the often-encountered difficulties in bacterial scFv expression.
authors.affiliation: Center for Genetic Engineering and Biotechnology, La Habana, Cuba.
authors.forename: M
authors.initials: M
authors.lastname: Ayala
authors.forename: R F
authors.initials: RF
authors.lastname: Balint
authors.forename: L
authors.initials: L
authors.lastname: Fernández-de-Cossío
authors.forename: J W
authors.initials: JW
authors.lastname: Canaán-Haden
authors.forename: J W
authors.initials: JW
authors.lastname: Larrick
authors.forename: J V
authors.initials: JV
authors.lastname: Gavilondo
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation; D021382:Protein Sorting Signals / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection / Q000235:genetics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D006514:Hepatitis B Surface Antigens; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C055966:immunoglobulin Fv