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In this chapter, protocols are described for converting mouse monoclonal antibodies into recombinant Fabs for transient expression in mammalian cells. Variable region genes are cloned by reverse transcription: PCR using either sequence specific or mixed 5' primers that hybridise to the first framework sequence of the mouse light and heavy chains and 3' primers that bind to the heavy- and light-chain constant regions. The amplified sequences are inserted into mammalian cell expression vectors by In-Fusion™ cloning. This method allows vector and amplified DNA sequences to be seamlessly joined in a ligation-independent reaction. Transient co-expression of light-chain and heavy-chain genes in HEK 293T cells enables production of recombinant Fabs for functional and structural studies.

Original publication

DOI

10.1007/978-1-61779-352-3_10

Type

Journal article

Journal

Methods Mol Biol

Publication Date

2012

Volume

801

Pages

137 - 159

Keywords

Amino Acid Sequence, Animals, Antibodies, Monoclonal, Base Sequence, Cloning, Molecular, DNA Primers, Enzyme-Linked Immunosorbent Assay, Gene Expression, Genetic Vectors, HEK293 Cells, Humans, Hybridomas, Immunoglobulin Fab Fragments, Immunoglobulin Heavy Chains, Immunoglobulin Light Chains, Mice, Molecular Sequence Data, Polymerase Chain Reaction, Protein Engineering, Recombinant Proteins, Transfection