کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
9902372 | 1545802 | 2005 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Ribosome display of antibodies: expression, specificity and recovery in a eukaryotic system
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
بیوتکنولوژی یا زیستفناوری
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Ribosome display of antibodies: expression, specificity and recovery in a eukaryotic system Ribosome display of antibodies: expression, specificity and recovery in a eukaryotic system](/preview/png/9902372.png)
چکیده انگلیسی
In ribosome display, proteins are linked to their encoding genetic material as protein-ribosome-mRNA complexes. The technology has been applied to the isolation of antibodies and other proteins from large PCR-derived libraries. Here we demonstrate the specificity of eukaryotic ribosome complexes and investigate recovery and display procedures using a single chain version of the anti-progesterone monoclonal antibody DB3. Complexes are formed by deletion of the 3â² stop codon in a coupled rabbit reticulocyte system. Using inhibition with different steroid probes, we show that the fine specificity of the combining site expressed as a nascent protein is closely similar to the native monoclonal, indicating correct folding and function while bound to the ribosome. We have demonstrated that the 3â² end of the mRNA is blocked by the stalled ribosome and unavailable to primers. Moreover, we show that an in situ RT-PCR recovery procedure, carried out on intact complexes, is more efficient than ribosome disruption and isolation of mRNA followed by RT-PCR. We also explore the Mg2+ and DTT concentrations and time required for efficient production of complexes. Our findings confirm the effectiveness of the eukaryotic ribosome display system and define conditions for efficient selection of single chain antibodies.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Immunological Methods - Volume 297, Issues 1â2, February 2005, Pages 73-82
Journal: Journal of Immunological Methods - Volume 297, Issues 1â2, February 2005, Pages 73-82
نویسندگان
Mingyue He, Michael J. Taussig,