Citation
- Authors: Fierle JK. et al.
- Year: 2019
- Journal: Sci Rep 9 12815
- Applications: in vitro / DNA / FectoPRO
- Cell type: HEK-293 6E
Description: Human embryonic kidney Fibroblast cell line genetically modified with a truncated version of EBNA1 which grows in suspension and chemically defined serum-free medium.
Method
- Recombinant protein was produced using the mammalian HEK293-6E/pTT transient expression system (National Research Council of Canada; obtained under licence).
- HEK293-6E cells were grown in Freestyle F17 medium (Thermo Fisher Scientific, #A13835) containing 4 mM GlutaMAX (Life Technologies, #35050061), 0.1% Pluronic® F-68 (Life Technologies, #24040032) and 25 μg/mL G418 (Life Technologies, #10131019) at 37 °C, 5% CO2 and 120 rpm.
- For transfection, the DNA was mixed with FectoPRO (Polyplus, #116-010) transfection reagent in F17 medium without supplements, according to the manufacturer’s instructions.
- After five days of protein expression, cultures were subjected to low speed centrifugation and the media collected.
- Samples could be used immediately for direct capture and immobilization (dCI) experiments or snap-frozen and stored at −80 °C until required.
Abstract
An early bottleneck in the rapid isolation of new antibody fragment binders using in vitro library approaches is the inertia encountered in acquiring and preparing soluble antigen fragments. In this report, we describe a simple, yet powerful strategy that exploits the properties of the SpyCatcher/SpyTag (SpyC/SpyT) covalent interaction to improve substantially the speed and efficiency in obtaining functional antibody clones of interest. We demonstrate that SpyC has broad utility as a protein-fusion tag partner in a eukaryotic expression/secretion context, retaining its functionality and permitting the direct, selective capture and immobilization of soluble antigen fusions using solid phase media coated with a synthetic modified SpyT peptide reagent. In addition, we show that the expressed SpyC-antigen format is highly compatible with downstream antibody phage display selection and screening procedures, requiring minimal post-expression handling with no sample modifications. To illustrate the potential of the approach, we have isolated several fully human germline scFvs that selectively recognize therapeutically relevant native cell surface tumor antigens in various in vitro cell-based assay contexts.
