Comparison of Escherichia coli and rabbit reticulocyte ribosome display systems.
Ribosome display is a technology for library selection and simultaneous molecular evolution in vitro. We present here a comparison between an optimized Escherichia coli system and different rabbit reticulocyte ribosome display systems, optimized in a number of parameters, as a coupled eukaryotic system had been suggested to result in high enrichment factors [He and Taussig (1997) Nucleic Acids Res. 25, 5132-5134]. With all systems, antibody scFv fragments, complexed to the ribosomes and the corresponding mRNA, were enriched by binding to their cognate antigen and enrichment was always dependent on the absence of a stop codon and the presence of cognate antigen. However, the efficiency of the E. coli ribosome display system was 100-fold higher than an optimized uncoupled rabbit reticulocyte ribosome display system, with separate in vitro transcription and translation, which was in turn several-fold more efficient than the reported coupled system. Neither the E. coli nor the rabbit reticulocyte ribosome display system was dependent on the orientation of the domains of an antibody scFv fragment or on the spacer sequence. In summary, we could not detect any intrinsic advantage of using a eukaryotic translation system for ribosome display.
Hanes
J
J
Biochemisches Institut der Universität Zürich, Switzerland.
Jermutus
L
L
Schaffitzel
C
C
Plückthun
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000032:analysis / Q000276:immunology; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014158:Transcription, Genetic
D000941:Antigens; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D010446:Peptide Fragments; D012333:RNA, Messenger; D016601:RNA-Binding Proteins