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A versatile bacteri...
A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination
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- Holmberg, Mats A. (författare)
- Stockholms universitet,Institutionen för molekylär biovetenskap, Wenner-Grens institut
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- Gowda, Naveen Kumar Chandappa (författare)
- Stockholms universitet,Institutionen för molekylär biovetenskap, Wenner-Grens institut
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- Andréasson, Claes, 1976- (författare)
- Stockholms universitet,Institutionen för molekylär biovetenskap, Wenner-Grens institut
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(creator_code:org_t)
- Elsevier BV, 2014
- 2014
- Engelska.
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 98, s. 38-45
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Abstract
Ämnesord
Stäng
- Production of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single-gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, we have developed an efficient cloning strategy based on yeast homologous recombination cloning (YHRC) into the new pET-based bacterial expression vector pSUMO-YHRC. The vector supports cloning for untagged expression as well as fusions to His6-SUMO or His6 tags. We demonstrate that YHRC from single PCR products of 6 independent genes into the vector results in virtually no background. Importantly, in a quantitative assay for functional expression we find that single-step YHRC of 7 DNA fragments can be performed with very high cloning efficiencies. The method and reagents described in this paper significantly simplifies the construction of expression plasmids from multiple DNA fragments, including complex gene fusions, chimeric genes and polycistronic constructs.
Ämnesord
- NATURVETENSKAP -- Biologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences (hsv//eng)
Nyckelord
- Cloning
- Nanobody
- Protein expression
- Protein purification
- Recombination cloning SUMO
- Cell Biology
- cellbiologi
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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