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Puri W cation of recombinant proteins from mammalian cell culture using a generic double-a Y nity chromatography scheme
| Content Provider | Semantic Scholar |
|---|---|
| Author | Cass, Brian Pham, Phuong L. Kamen, Amine A. Durocher, Yves |
| Copyright Year | 2004 |
| Abstract | Transient 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 eYcient puriWcation scheme that can be applied generically, typically through the use of aYnity tags such as the polyhistidine-tag for capture by immobilized metal-aYnity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin aYnity ligand. However, one-step puriWcation 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 eYcient puriWcation by consecutive IMAC and StrepTactin aYnity. 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 puriWed to >99% homogeneity with yields varying from 29 to 81%. Crown copyright 2004 Published by Elsevier Inc. All rights reserved. |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | http://sciencedirect.b2b.ntent.com/ERA/ResourceHandler.ashx?2b3d75f3-a246-41d7-9db9-ee4ba190bfcb%3BItem+4,+Cass+et+al.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
