Converting Monoclonal Antibodies into Fab Fragments for Transient Expression in Mammalian Cells

Abstract

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.

The role of the light chain in the structure and binding activity of two cattle antibodies that neutralize bovine respiratory syncytial virus

“…The CH1 heavy chain is Bos taurus IgG1 (Genbank S82409: (Kacskovics and Butler, 1996)). Synthetic genes encoding the constant regions were inserted by Infusion ® cloning into PmeI-HindIII cut pOPING-ET (Nettleship et al, 2008b, 2012) The vectors have been engineered so that VH and VL sequences can be inserted into the KpnI-PstI (pOPINBOVL) and KpnI-SfoI (pOPINBOVH) restriction sites by Infusion ® cloning. Synthetic genes encoding the candidate variable regions (B4 and B13 VH and VL) (Armour et al, 1994) were purchased from IDT Technology as “Infusion-ready” gBlocks and inserted into the pOPIN expression vectors.…”

Generation and characterisation of recombinant FMDV antibodies: Applications for advancing diagnostic and laboratory assays

“…The CH1 heavy chain is Bos taurus IgG1 (Genbank S82409). Synthetic genes encoding the constant regions were inserted by In-Fusion ® cloning (Clontech) into PmeI-HindIII cut pOPING-ET [ 56 , 57 ]. The VH and VL sequences were inserted into the KpnI-PstI (pOPINBOVH) and KpnI-SfoI (pOPINBOVL) restriction sites respectively by In-Fusion ® cloning.…”

Overview of a High-Throughput Pipeline for Streamlining the Production of Recombinant Proteins

“…The mass spectrum for OPPF 20371 + 18732 shows one peak corresponding to a non-reduced Fab fragment where the heavy and light chains are linked by a cysteine bridge. This was produced by co-transfection in mammalian cells 14 [18]. In the case of OPPF 3325, although purified protein could be seen by SDS-PAGE, the protein failed quality assurance by mass spectrometry (Fig.…”