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Production of a Thermostable DNA Polymerase by Site-Specific Cleavage of a Heat-Eluted Affinity Fusion Protein
KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.ORCID-id: 0000-0002-5391-600X
KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.ORCID-id: 0000-0001-8993-048X
Vise andre og tillknytning
1997 (engelsk)Inngår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 9, s. 125-132Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
1997. Vol. 9, s. 125-132
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Identifikatorer
URN: urn:nbn:se:kth:diva-13273DOI: 10.1006/prep.1996.0674OAI: oai:DiVA.org:kth-13273DiVA, id: diva2:323028
Merknad
QC 20100609Tilgjengelig fra: 2010-06-09 Laget: 2010-06-09 Sist oppdatert: 2017-12-12bibliografisk kontrollert
Inngår i avhandling
1. Protein Engineering by Directed Evolution and Rational Design
Åpne denne publikasjonen i ny fane eller vindu >>Protein Engineering by Directed Evolution and Rational Design
2001 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
sted, utgiver, år, opplag, sider
Stockholm: KTH, 2001. s. 87
Emneord
ion-exchange chromatography, subtilisin, Klenow DNA polymerase, Taq DNA polymerase, directed evolution, rational design, charge engineering, phosphonylating inhibitor, phage display, circular dichroism, protein A
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-3171 (URN)91-7283-102-2 (ISBN)
Disputas
2001-06-01, 00:00
Merknad
QC 20100609Tilgjengelig fra: 2001-05-31 Laget: 2001-05-31 Sist oppdatert: 2010-06-09bibliografisk kontrollert

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Gräslund, TorbjörnUhlén, MathiasNygren, Per-Åke

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