Whole-Gene Mutagenesis Libraries for Antibody Engineering

The BG505 SOSIP whole-gene saturation mutagenesis and “rare” amino acid libraries were synthesized by Integrated DNA Technologies and GenScript, respectively. Libraries for germline targeting were created by error-prone PCR (GeneMorph II, Agilent), site-directed mutagenesis (QuikChange, Agilent) or two-step assembly PCR of degenerate primers with the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and cloned into a modified version of the gateway cloning entry vector pENTR/D-TOPO (Ota et al., 2012 (link)) with the circular polymerase extension cloning (CPEC) method (Quan and Tian, 2014 (link)) or Gibson Assembly (New England Biolabs), according to the manufacturer’s instructions. All libraries were then transferred to the lentiviral vector pLenti CMVTRE3G puro Dest (Ota et al., 2012 (link)) with the LR Clonase II enzyme mix (Thermo Scientific).