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

  • Joanne E. Nettleship
  • Aleksandra Flanagan
  • Nahid Rahman-Huq
  • Rebecca Hamer
  • Raymond J. OwensEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 801)


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.

Key words

HEK293T PCR cloning Fabs 



The Oxford Protein Production Facility is supported by grants from the Medical Research Council, UK, the Biotechnology and Biological Sciences Research Council, UK. AF is the recipient of a Wellcome Trust Research Studentship.


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Joanne E. Nettleship
    • 1
  • Aleksandra Flanagan
    • 2
  • Nahid Rahman-Huq
    • 3
  • Rebecca Hamer
    • 4
  • Raymond J. Owens
    • 3
    Email author
  1. 1.Oxford Protein Production Facility UKResearch Complex at Harwell, Rutherford Appleton LaboratoryOxfordshireUK
  2. 2.Division of Structural BiologyWellcome Trust Centre for Human GeneticsOxfordUK
  3. 3.Oxford Protein Production Facility UKResearch Complex at Harwell, Rutherford Appleton LaboratoryOxfordshireUK
  4. 4.Department of StatisticsUniversity of OxfordOxfordUK

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