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.

Appendix

The nucleotide sequences of all of the known mouse antibody heavy (VH) and light-chain (VL) variable domains are grouped into a series of templates (5). Variation within each template is shown by the wobble codons. For each template, the number of different variants and hence the number of different primers required to amplify the sequences in the template is given (d). The occurrence of any given template in mouse antibody sequences is shown as a percentage (%rep). For both VH and VL sequences, an example is given of using a filter to limit the total number of primers needed to amplify most of the sequences. Thus, by eliminating VH and VL sequences with high variability and low representation, the total number of unique primers required can be reduced to 43 unique primers for VH and 116 unique primers for VL, which cover 87 and 83% of known mouse VH and VL sequences, respectively. The Infusion cloning tags shown in Table 1 are added to the 5′ end of each primer. The primers are synthesised individually to ensure equal representation of each sequence in the final pooled primer sets.