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Selection of Single-Chain Antibody Fragments (scFv) by Ribosome Display

Ribosome display can be used to select single-chain antibody fragments against a variety of targets. This is the most widely used application for ribosome display. Creative Biolabs provides both prokaryotic ribosome display system and eukaryotic ribosome display system for scFv selection.

Ribosome display is a powerful cell-free technology for the selection and evolution of proteins. Ribosome display is based on in vitro translation and transcription which can couple phenotype (DNA and RNA) and genotype (protein). Newly synthesized protein and the mRNA encoding are preserved on the ribosome to form stable protein-ribosome-mRNA (PRM) complexes. Since no cells need to be transformed, this technique avoids the limitation of library size, and very large libraries can be used directly in selections. Besides, the in vitro amplification provides a very convenient ways for direct mutagenesis for protein evolution. Moreover, ribosome display is suitable for generating toxic, proteolytically sensitive and unstable proteins.

Selection of Single-Chain Antibody Fragments (scFv)

Creative Biolabs has developed ribosome display system to carry out phenotypic selection for scFv against a variety of peptides and proteins targets. During this process, sequence evolution and screening can be performed simultaneously. We select picomolar affinity antibody fragments from various in vitro antibody libraries [immunized antibody library (e.g. mouse immunized library), naïve antibody library, semisynthetic antibody library, and totally synthetic antibody library] against peptides and proteins. We can easily construct a ribosome display library which contains 1012 or more independent functional members after each randomization step and used for affinity selection.

Construction of VH/K library, starting from spleen cell mRNA. ‘X’ indicates that the 3’ stop codon has been deleted; shaded rectangle represents the linker. T7Ab/back and Ck/for are primers. Fig. 1. Construction of VH/K library, starting from spleen cell mRNA. ‘X’ indicates that the 3’ stop codon has been deleted; shaded rectangle represents the linker. T7Ab/back and Ck/for are primers. (He et al. 1999)

Protein evolution (mutation) can occur during selection when DNA polymerases without proofreading function are used in PCR. Other methods, such as DNA shuffling, error-prone PCR, or the staggered extension process can be used to increase the diversity of the proteins. Ribosome display is a very powerful method for the directed evolution of proteins and selection of the best scFv to meet all your requirements.

Creative Biolabs can provide both the prokaryotic ribosome display system and the eukaryotic ribosome display system. Prokaryotic system using E. coli S30 is simple and easy to perform while a eukaryotic system using rabbit reticulocyte lysate is more efficiency, ease of manipulation and sensitivity of recovery.

The proteins we enriched by ribosome display can be produced in vivo or in vitro to yield reasonable amounts of protein for further analysis.

For more detailed information, please feel free to contact us or directly sent us an inquiry.

References

  1. He M and Taussig M J (2002) “Ribosome display: cell-free protein display technology.” Brief Funct Genomic Proteomic 1(2):204-212.
  2. He M, Edwards B M, Kastelic D, et al. (2012) “Eukaryotic ribosome display with in situ DNA recovery.” Methods Mol Biol 805:75-85.
  3. He M, Menges M, Groves M A, et al. (1999) “Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display.” J Immunol Methods 231(1-2):105-117.



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