The wt hAID enzyme and selected mutants were obtained in a bacterial expression system (see Methods).
Through site-specific mutagenesis, several improved mutants were obtained, with GadB1E312S being the best one.
SxVis (ExoS233 409 fused to Vis218 249) and the ExoS233 453 mutants were obtained as synthetic clones, sub-cloned in pET151b (GeneArt/ThermoFischer Scientific).
Via this method, four new fluorescent protein mutants were obtained based on monomeric cherry fluorescent protein.
The following mutants were obtained: Asp170Glu, Gly178Ser, Leu218Ser or Thr, Arg238Val, Thr245Ala or Ser and Ser264Lys or Ile.
By screening 400 clones from random mutant libraries for enhanced fibrinolytic activity, two mutants were obtained: N111R, R230G.
These mutants were obtained through evolutionary modifications in two specific regions of the CPMO's putative active site.
Recombinant proteins including PML∆BC (i.e., the deletion of residues, 120 360) and mutants were obtained by in vitro translation (Promega) using pcDNA3.1 vector with T7 promoter according to manufacturer's standard protocol.
Twenty-eight azoxystrobin-resistant mutants were obtained by UV mutagenesis and subsequent selection on medium amended with azoxystrobin (12 µg/ml) and salicylhydroxamic acid.
HLA-A2 antigen mutants were obtained previously from the B lymphoblastoid cell line T5-1 by mutagenesis followed by immunoselection.
The osphyA osphyB double mutants were obtained by crossing osphyA-2 and osphyB-2 single mutants.
