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Rezensionsexemplar
Oktober 2014
Alexander Maaß
Evolutive Ansätze zur Affinitätsmaturierung von Antikörper-Fragmenten durch das immunrelevante Protein Activation-induced Cytidine Deaminase
The scope of this thesis was the in vivo and in vitro mediated maturation of antibodies by the protein AID. By simultaneous maturation and screening in each selection round it was possible to isolate affinity-optimized single clones against specific targets.
His-AID731 was successfully produced recombinantly and the activity was detected. Single- and double strand DNA which coded for thinly affinity shark-antibodies was treated with AID enzyme. The resulting mixture of Uracil-containing DNA was amplified via PCR and the DNA mutant-mixture was integrated in yeast cells. The resulting library was sorted for binding proteins with increased affinity against the target protein by high-throughput screening and affinity-optimized antibodies were isolated against the target protein in selection round two and three by yeast display. This evolutionary affinity maturation of antibodies fragments by the human activation-induced cytidine deaminase (AID) indicates that the antibody optimization from human B cells could be imitated in the S. cerevisiae.
His-AID731 was successfully produced recombinantly and the activity was detected. Single- and double strand DNA which coded for thinly affinity shark-antibodies was treated with AID enzyme. The resulting mixture of Uracil-containing DNA was amplified via PCR and the DNA mutant-mixture was integrated in yeast cells. The resulting library was sorted for binding proteins with increased affinity against the target protein by high-throughput screening and affinity-optimized antibodies were isolated against the target protein in selection round two and three by yeast display. This evolutionary affinity maturation of antibodies fragments by the human activation-induced cytidine deaminase (AID) indicates that the antibody optimization from human B cells could be imitated in the S. cerevisiae.
Schlagwörter: AID; Antikörper; Yeast; Affinitätsmaturierung; Activation-induced Cytidine Deaminase
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