کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1988634 1063325 2006 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Preparation and characterization of scFv for affinity purification of reteplase
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Preparation and characterization of scFv for affinity purification of reteplase
چکیده انگلیسی

The work was to explore the feasibility of protein affinity purification using ligand isolated from phage library. Reteplase was used as the model protein and a humanized semi-synthetic single chain fragment variable phage library as the source of ligand. After four rounds of biopanning, reteplase-specific phage clones were greatly enriched. The scFv gene from the best phage clone was inserted to pET-29a and expressed in E. coli Rosseta. After purification by nickel-affinity and refolding, this scFv protein was proven to recognize reteplase specifically and sensitively in ELISA and dot-blotting. Its binding constant to reteplase was 1.84 × 10− 8 M, measured by surface plasmon resonance. After immobilized on Sepharose 4B, the scFv was used for the affinity purification of reteplase from milk. It was found that reteplase was highly purified from the starting material. In conclusion, it has been demonstrated that humanized scFv prepared with this approach could be used as a practical affinity ligand for efficient and cost-effective purification of reteplase, as well as other therapeutic proteins.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Biochemical and Biophysical Methods - Volume 67, Issue 1, 30 April 2006, Pages 27–36
نویسندگان
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