Comparison of Escherichia coli and rabbit reticulocyte ribosome display systems

FEBS Letters

Schaffitzel, Christiane¹  Hanes, Jozef¹  Jermutus, Lutz¹  Plückthun, Andreas¹ 

[1] Biochemisches Institut der Universität Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland

关键词: Ribosome display;    In vitro translation;    Library;    Directed evolution;    scFv;    single-chain Fv fragment of an antibody in which the VL domain is linked to the VH domain by a linker;    VL;    variable domain of the light chain;    VH;    variable domain of the heavy chain;   

DOI  :  10.1016/S0014-5793(99)00475-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown