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PUREfrex® 2.1 / DsbC Set / DnaK Mix / cell-free IgG synthesis / Trastuzumab / IgG1 / IgG2 / IgG4
Aglycosylated IgG (including IgG1, IgG2 and IgG4 subclasses) were synthesized using PUREfrex®. The PURE (Protein synthesis Using Recombinant Elements) system is a reconstituted cell-free protein synthesis system, which consists of only purified factors necessary for transcription, translation and energy regeneration. Recently, we developed an updated PURE system with higher productivity, which was launched as “PUREfrex® 2.0” in 2015. We reported that PUREfrex® 2.0 could be used for production of fragment antibodies such as Fab and scFv by last year.
Here, we report the further application using PUREfrex® 2.0 for production of IgG. We chose Trastuzumab as a model protein and optimized the composition of PUREfrex® 2.0 reaction mixture and the reaction conditions as below; 1) adding disulfide bond isomerase (DsbC); 2) choosing GSH as an effective reductant; 3) adjusting GSH/GSSG ratio; 4) adding molecular chaperone (DnaK mix); 5) long-time incubation for 28h. We also suggested that synthesis temperature and template DNA ratio (light chain/heavy chain) should be optimized for individual IgGs for the best yield. At the best mode of synthesis, the productivity of Trastuzumab reached to 124 µg/mL. Moreover, the 68 µg of purified Trastuzumab was obtained from 1 mL of the reaction mixture after the purification by protein A resin and the following gel filtration. The purified Trastuzumab exhibited high binding affinity to recombinant HER2 protein (KD=4.24E-10 M) and internalized into HER2 expressing BT-474 cells. Furthermore, other IgGs including IgG1, IgG2 and IgG4 subclasses were also synthesized and confirmed their binding activity in ELISA.
These results indicate that PUREfrex® will be useful tool for high-throughput production/screening of functional antibodies (scFv, Fab, and IgG).