Protein affinity maturation in vivo using E. coli mutator cells.
This protocol provides a simple in vivo strategy for introducing random mutations to a target DNA sequences using E. coli mutator cells. The method has been used in our laboratory for affinity maturation of proteins encoded by target DNA sequences. Selection conditions can be modified for antibody fragments with increased production levels. Growth conditions in E. coli mutator cells can be adjusted to introduce a single random point mutation per kilobase of DNA, approximately equivalent to one codon change per scFv fragment.
Coia
G
G
CRC for Diagnostic Technologies, CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville, Vic. 3052, Australia. greg.coia@hsn.csiro.au
Hudson
P J
PJ
Irving
R A
RA
D000906:Antibodies / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D019151:Peptide Library; D017354:Point Mutation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D004274:DNA, Recombinant; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; C055966:immunoglobulin Fv