Reprint of: Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme

Download
  1. Get@NRC: Reprint of: Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme (Opens in a new window)
DOIResolve DOI: http://doi.org/10.1016/j.pep.2011.08.012
AuthorSearch for: ; Search for: ; Search for: ; Search for:
TypeArticle
Journal titleProtein Expression and Purification
ISSN1046-5928
SubjectTransient transfection; Human embryonic kidney cells; Immobilized metal-affinity chromatography; His-tag; Strep-tag II; StrepTactin affinity chromatography
AbstractTransient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His) 8 to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%. Crown Copyright © 2004.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); NRC Biotechnology Research Institute
Peer reviewedYes
NPARC number21271516
Export citationExport as RIS
Report a correctionReport a correction
Record identifier1af41c1b-06e9-448b-84b4-9e104fba8df7
Record created2014-03-24
Record modified2016-05-09
Bookmark and share
  • Share this page with Facebook (Opens in a new window)
  • Share this page with Twitter (Opens in a new window)
  • Share this page with Google+ (Opens in a new window)
  • Share this page with Delicious (Opens in a new window)
Date modified: