Cloning and Expression of Antibody Variable Regions

Heavy and light chain variable regions were cloned in pVitro-hygro-1 (Invivogen) using polymerase incomplete primer extension (PIPE) cloning as previously described (25 (link)). In brief, pVitro-hygro-1, containing pre-cloned human antibody heavy and light constant region cassettes (gamma 1/kappa) was used as a template in two separate PIPE PCR reactions to amplify two linear plasmid fragments with partially single-stranded 5″ ends. Similarly, the Ig heavy and light chain variable regions were PIPE PCR-amplified, using the pCR-Blunt constructs, generated previously, as PCR templates. Next, the PCR products were diluted four times with ddH₂O and mixed in a ratio of 1:1:1:1, incubated at RT for 1 h, and 10 µl of the mixture were used to transform Top10 OneShot™ E. coli cells (Thermo Fisher Scientific). Successful cloning was confirmed by Sanger sequencing (Source BioScience). MOv18 IgG1/k was expressed transiently in Expi293F™ cells (Thermo Fisher Scientific), as described in Ref. (26 (link)). Antibodies, secreted in the Expi293F™ culture supernatant, were purified using a Protein A column (Thermo Fisher Scientific) and stored in PBS at 4°C.