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A pseudoknot improves selection efficiency in ribosome display.

Authors
Kim, JM; Shin, HJ; Kim, K; Lee, MS
Citation
Molecular biotechnology, 36(1):32-37, 2007
Journal Title
Molecular biotechnology
ISSN
1073-60851559-0305
Abstract
The size and diversity of ribosome display libraries depends upon stability of the complex formed between the ribosome, mRNA and translated protein. To investigate if mRNA secondary structure improves stability of the complex, we tested a pseudoknot, originating from the genomic RNA of infectious bronchitis virus (IBV), a member of the positive-stranded coronavirus group. We used the previously-isolated anti-DNA scFv, 3D8, as a target protein. During in vitro translation in rabbit reticulocyte lysate, we observed that incorporation of the pseudoknot into the mRNA resulted in production of a translational intermediate that corresponded to the expected size for ribosomal arrest at the pseudoknot. Complexes containing the mRNA pseudoknot exhibited a higher efficiency of affinity selection than that those without, indicating that the pseudoknot improves stability of the mRNA-ribosome-antibody complex in a eukaryotic translation system. Thus, in order to improve the efficiency of selection, this relatively short pseudoknot sequence could be incorporated into ribosome display.
MeSH terms
Amino Acid SequenceAnimalsBase SequenceDNA/geneticsImmunoglobulin Variable Region/chemistryImmunoglobulin Variable Region/geneticsInfectious bronchitis virus/chemistryInfectious bronchitis virus/geneticsMiceMolecular Sequence DataNucleic Acid Conformation*PlasmidsProtein BindingProtein BiosynthesisRNA, Messenger/geneticsRNA, Messenger/metabolismRNA, Viral/chemistry*RNA, Viral/geneticsRabbitsRibosomes/metabolism*
PMID
17827535
Appears in Collections:
Journal Papers > School of Medicine / Graduate School of Medicine > Microbiology
AJOU Authors
신, 호준김, 경민
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