• Authors: Ereño-Orbea J. et al.
  • Year: 2018
  • Journal: J Mol Biol 430 322-336
  • Applications: in vitro / DNA / FectoPRO
  • Cell types:
    1. Name: HEK-293F
    2. Name: HEK-293S


Expression and purification of Fabs: - The heavy and light chains of Fabs were synthesized by GeneArt and subcloned into the pHLsec expression vector. To enhance crystallizability, the predicted N-linked glycosylation site at position 58 in pinatuzumab Fab HC was mutated to a glutamine. - HEK293F cells were transfected with heavy and light chain plasmids (1:0.5 ratio) in a total DNA amount of 90 ug for each 200 mL transfection. FectoPRO (Polyplus Transfections) was used as a transfection reagent in a 1:1 ratio of DNA:FectoPRO. - Cells were transfected at a cell density of 0.8 x 106 cells mL-1 and incubated at 37oC, 125 rpm, 8% CO2 in an Multitron Pro for 5-7 days. Expression and purification of CD1920-291 and CD2220-687: - Full-length human CD19 ectodomain (ECD) (CD1920-291) fused to an expression protein bearing a His6x tag was codonoptimized for expression in human cells and synthesized by GeneArt. - The CD1920-291 construct was transiently transfected into HEK293 Gnt I-/- (HEK293S) suspension cells. Cells were split in 200 mL cultures at 0.8 x 106 cells mL-1. 50 µg of DNA was filtered and mixed in a 1:1 ratio with transfection reagent FectoPRO (Polyplus Transfections), and incubated at room temperature for 10 min. - The DNA:FectoPRO solution was then added directly to the cells, and cells were incubated at 37oC, 180 rpm, 8% CO2 in a Multitron Pro shaker (Infors HT) for 6-7 days.


Monoclonal antibodies constitute one of the largest groups of drugs to treat cancers and immune disorders, and are guiding the design of vaccines against infectious diseases. Fragments antigen-binding (Fabs) have been preferred over monoclonal antibodies for the structural characterization of antibody-antigen complexes due to their relatively low flexibility. Nonetheless, Fabs often remain challenging to crystallize because of the surface characteristics of complementary determining regions and the residual flexibility in the hinge region between the variable and constant domains. Here, we used a variable heavy-chain (VHH) domain specific for the human kappa light chain to assist in the structure determination of three therapeutic Fabs that were recalcitrant to crystallization on their own. We show that this ligand alters the surface properties of the antibody-ligand complex and lowers its aggregation temperature to favor crystallization. The VHH crystallization chaperone also restricts the flexible hinge of Fabs to a narrow range of angles, and so independently of the variable region. Our findings contribute a valuable approach to antibody structure determination and provide biophysical insight into the principles that govern the crystallization of macromolecules.