Skip to main content
. 2017 Sep 11;8:1112. doi: 10.3389/fimmu.2017.01112

Figure 1.

Figure 1

Schematic representation of the pipeline for generation and production of wild-type (WT) and Fc mutant IgG antibodies. (A) WT antibody construct in pVitro1-hygro-mcs. (B) Polymerase incomplete primer extension (PIPE) PCR linearization and mutagenesis of the WT construct to generate pVitro1 DNA fragments carrying the N297Q (left, fragments 1 and 4) or S239D/I332E (right, fragments 1, 2, and 4) mutations. Mutations indicated by “*”. (C) Introduction of mutations in WT constructs through mutagenic PIPE primers. (D) DpnI digestion. (E) Enzyme-independent assembly of the linear pVitro1 fragments. (F) Bacterial transformation of the assembled constructs. (G) Confirmation of the insertion of desired mutations. (H,I) Recombinant expression in Expi293F cells (H) and purification (I) of antibody WT and mutant variants.