Engineering a recyclable elastin-like polypeptide capturing scaffold for non-chromatographic protein purification.

Publisher: Wiley-Blackwell Country of Publication: United States NLM ID: 8506292 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1520-6033 (Electronic) Linking ISSN: 15206033 NLM ISO Abbreviation: Biotechnol Prog Subsets: MEDLINE

Publication: <2010-> : Hoboken, NJ : Wiley-Blackwell
Original Publication: [New York, N.Y. : American Institute of Chemical Engineers, c1985-

Biotechnology* ; Protein Engineering*; Elastin/*isolation & purification ; Peptides/*isolation & purification; Elastin/genetics ; Electrophoresis, Polyacrylamide Gel ; Peptides/genetics ; Polymerase Chain Reaction ; Protein Structure, Tertiary ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification

Previously, we reported a non-chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium thermocellum and the reversible aggregation property of elastin-like polypeptide (ELP) to provide fast and cost-effective protein purification. However, the bound dockerin-intein tag cannot be completely dissociated from the ELP-cohesin capturing scaffold due to the high binding affinity, resulting in a single-use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium-coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA-mediated dissociation of the bound dockerin-intein tag from the ELP-cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non-chromatographic based affinity method provides an attractive approach for efficient and cost-effective protein purification.
(© 2013 American Institute of Chemical Engineers.)

Keywords: cohesin-dockerin; elastin-like polypeptide; intein; protein purification; recycle

0 (Peptides)
0 (Recombinant Proteins)
9007-58-3 (Elastin)

Date Created: 20130627 Date Completed: 20140514 Latest Revision: 20130812

20250114

10.1002/btpr.1757

23801586