Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris.
We have applied the combinatorial immunoglobulin library and phage display technologies to generate monoclonal rabbit single-chain Fv (scFv) antibody fragments specific for recombinant human leukemia inhibitory factor (rhLIF). The B cell immunoglobulin repertoire of an immunized rabbit was immortalized by the combinatorial cloning of the rearranged variable domains of light (VL) and heavy (VH) chains. Affinity selection of the library displaying the rabbit antibody domains on the phage surface resulted in the isolation of phage encoding scFv antibodies which specifically bind to the antigen. We utilized the methylotrophic yeast Pichia pastoris for high level secretion of soluble and functional scFv antibody fragment. More than 100 mg/L of pure and functional rabbit anti-rhLIF scFv antibody was obtained directly from the P. pastoris culture supernatant by one-step affinity chromatography.
Ridder
R
R
Sandoz Pharma, Department of Drug Metabolism and Pharmacokinetics, Basel, Switzerland.
Schmitz
R
R
Legay
F
F
Gram
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D015723:Gene Library; D006131:Growth Inhibitors / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines / Q000276:immunology; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D006131:Growth Inhibitors; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D015850:Interleukin-6; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines; D011994:Recombinant Proteins