Rapid discovery and optimization of therapeutic antibodies against emerging infectious diseases

Rogers, J.; Schoepp, R.J.; Schröder, O.; Clements, T.L.; Holland, T.F.; Li, J.Q.; Li, J.; Lewis, L.M.; Dirmeier, R.P.; Frey, G.J.; Tan, X.; Wong, K.; Woodnutt, G.; Keller, M.; Reed, D.S.; Kimmel, B.E.; Tozer, E.C.

Light-and heavy-chain PCR products were fused by overlapping PCR. Antibody light and heavy chains were cloned into NdeI and PacI restriction sites to form pBAD_Fab_ZF. The cassette contained a nucleotide sequence encoding a light chain antibody domain, a non-translated linker, and a heavy chain antibody domain fused in-frame to the N-terminus of the zinc finger protein. The light and heavy chain/zinc finger proteins were expressed as separate polypeptide chains. The pBAD_Fab_ZF was transformed into high efficiency XL1-Blue cells, plasmids were isolated, and subsequently transformed into Rosettagami TM (DE3) competent cells (Novagen, Madison, WI, USA). A library stock of these transformed expression host cells was stored in 10% glycerol at 2708C.