A tool kit for rapid cloning and expression of recombinant antibodies

TY - JOUR

T1 - A tool kit for rapid cloning and expression of recombinant antibodies

AU - Dodev, Tihomir S

AU - Karagiannis, Panagiotis

AU - Gilbert, Amy E

AU - Josephs, Debra H

AU - Bowen, Holly

AU - James, Louisa K

AU - Bax, Heather J

AU - Beavil, Rebecca

AU - Pang, Marie O

AU - Gould, Hannah J

AU - Karagiannis, Sophia N

AU - Beavil, Andrew J

PY - 2014/7/30

Y1 - 2014/7/30

N2 - Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.

AB - Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.

KW - Animals

KW - Antigens, Plant/biosynthesis

KW - Base Sequence

KW - Calcium-Binding Proteins/biosynthesis

KW - Cloning, Molecular/methods

KW - DNA Primers/chemical synthesis

KW - Epitopes/chemistry

KW - Escherichia coli/genetics

KW - Gene Expression

KW - Genetic Vectors/chemistry

KW - Humans

KW - Immunoglobulin E/biosynthesis

KW - Immunoglobulin G/biosynthesis

KW - Immunoglobulin kappa-Chains/biosynthesis

KW - Immunoglobulin lambda-Chains/biosynthesis

KW - Molecular Sequence Data

KW - Plant Proteins/biosynthesis

KW - Polymerase Chain Reaction

KW - Reagent Kits, Diagnostic

KW - Recombinant Fusion Proteins/biosynthesis

U2 - 10.1038/srep05885

DO - 10.1038/srep05885

M3 - SCORING: Journal article

C2 - 25073855

VL - 4

SP - 5885

JO - SCI REP-UK

JF - SCI REP-UK

SN - 2045-2322

ER -