Fig. 2.
Expression and purification of Hu-E16 mAb in N. benthamiana plants. N. benthamiana leaves were coinfiltrated with Hu-E16 LC and HC constructs. Leaf proteins were extracted on days 4–10 after agroinfiltration (A) or on day 7 after agroinfiltration (B). (A) Protein extracts were analyzed with an ELISA that detects the assembled form of pHu-E16 mAb. Mean ± SD of samples from three independent infiltration experiments are presented. (B) Leaf protein extract was purified and analyzed on a 4–20% SDS-PAGE gel under a reducing (lanes 1–6) or nonreducing (lanes 7 and 8) condition. Lane 1, clarified plant extract; lane 2, plant proteins removed by 25% ammonium sulfate precipitation; lane 3, 50% ammonium sulfate pellet fraction resuspended for protein A chromatography; lane 4, protein A flow-through fraction; lanes 5 and 7, purified pHu-E16 mAb in the protein A eluate; lanes 6 and 8, mHu-E16 as a reference standard. ◂: RuBisCo large and small subunits;←: LC, HC, and assembled form (HL)2 of Hu-E16 mAb.