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Production of a Thermostable DNA Polymerase by Site-Specific Cleavage of a Heat-Eluted Affinity Fusion Protein
KTH, Superseded Departments, Biochemistry and Biotechnology.ORCID iD: 0000-0002-5391-600X
KTH, Superseded Departments, Biochemistry and Biotechnology.
KTH, Superseded Departments, Biochemistry and Biotechnology.ORCID iD: 0000-0001-8993-048X
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1997 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 9, 125-132 p.Article in journal (Refereed) Published
Abstract [en]

A novel strategy is described for bacterial expression and affinity purification of a recombinant truncated version of the heat-stable DNA polymerase I fromThermus aquaticus.The DNA polymerase ([Delta]Taq) was produced as a fusion to a serum albumin binding affinity handle (ABP) derived from streptococcal protein G. Based on the thermostability of the [Delta]TaqDNA polymerase, affinity-purified ABP-[Delta]Taqcould be heat-eluted from HSA columns by incubation at 85ï¿œC. To produce free [Delta]TaqDNA polymerase, efficient site-specific cleavage of the affinity tag was performed using a recombinant coxsackievirus 3C protease (3Cpro), also produced as an ABP affinity fusion. Thus, an integrated strategy could be devised where both the cleaved ABP affinity tag and the protease fusion could be recovered after site-specific cleavage using HSA-affinity chromatography. The flow-through fraction contained essentially pure [Delta]TaqDNA polymerase with full enzymatic activity.

Place, publisher, year, edition, pages
1997. Vol. 9, 125-132 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-13273DOI: 10.1006/prep.1996.0674OAI: oai:DiVA.org:kth-13273DiVA: diva2:323028
Note
QC 20100609Available from: 2010-06-09 Created: 2010-06-09 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Protein Engineering by Directed Evolution and Rational Design
Open this publication in new window or tab >>Protein Engineering by Directed Evolution and Rational Design
2001 (English)Doctoral thesis, comprehensive summary (Other scientific)
Place, publisher, year, edition, pages
Stockholm: KTH, 2001. 87 p.
Keyword
ion-exchange chromatography, subtilisin, Klenow DNA polymerase, Taq DNA polymerase, directed evolution, rational design, charge engineering, phosphonylating inhibitor, phage display, circular dichroism, protein A
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-3171 (URN)91-7283-102-2 (ISBN)
Public defence
2001-06-01, 00:00
Note
QC 20100609Available from: 2001-05-31 Created: 2001-05-31 Last updated: 2010-06-09Bibliographically approved

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Publisher's full texthttp://www.sciencedirect.com/science/article/B6WPJ-45KKW49-42/2/03c036fd1bf934b0f3d5fd8d3c0995f3

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Gräslund, TorbjörnUhlén, MathiasNygren, Per-Åke

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