Abstract
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 1.84x10(-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.
References
Feb 1, 1991·Bio/technology·F H Arnold
Jul 18, 1985·Biochimica Et Biophysica Acta·M EinarssonL Kaplan
Nov 15, 1981·Thrombosis Research·C A EggletonD P Thomas
Jul 15, 1994·The EMBO Journal·A D GriffithsT J Allison
Jan 1, 1996·Archives of Biochemistry and Biophysics·S ZhaoE Y Lee
Aug 6, 1996·Proceedings of the National Academy of Sciences of the United States of America·H BiersackA H Andersen
Dec 19, 1996·The American Journal of Cardiology·C BodeW Kübler
Dec 1, 1996·Molecular Biotechnology·M A Vijayalakshmi
Oct 1, 1996·Journal of Christian Nursing : a Quarterly Publication of Nurses Christian Fellowship·K Wagoner
Jul 14, 1998·Immunotechnology : an International Journal of Immunological Engineering·H R HoogenboomR C Roovers
Jul 16, 1999·Journal of Biotechnology·L SánchezJ V Gavilondo
Feb 11, 2000·Biotechnology and Applied Biochemistry·L GuoG Meng
Aug 1, 2000·Immunology Today·M Clark
Oct 19, 2002·Transfusion·Johannes BlümelJohannes Löwer
May 28, 2003·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·N E Labrou
Oct 11, 2003·Biotechnology Advances·I Benhar
May 24, 2005·Protein and Peptide Letters·Martin LinhultSophia Hober
Aug 10, 2005·APMIS : Acta Pathologica, Microbiologica, Et Immunologica Scandinavica·Louise LohseLis Eriksen
Oct 12, 2011·Journal of Immunological Methods·Theo RispensLucien A Aarden
Citations
Jun 3, 2015·Biotechnology and Bioengineering·Conor FieldsGil U Lee
Dec 13, 2007·Journal of Molecular Recognition : JMR·Rebecca L Rich, David G Myszka
Dec 29, 2011·Epidemiology and Psychiatric Sciences·F LindbladA Hjern
Dec 15, 2012·Journal of Attention Disorders·Claudia E van der PutGeert Jan J M Stams
Oct 24, 2013·Journal of Attention Disorders·Ana MirandaCarla Colomer
Dec 9, 2015·The Australian and New Zealand Journal of Psychiatry·Gin S MalhiAjeet B Singh
Jun 15, 2019·Bioscience Reports·Rui LuYong Cheng