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Overexpression, rap...
Overexpression, rapid isolation, and biochemical characterization of Escherichia coli single-stranded DNA-binding protein
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Ehn, M. (författare)
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- Nilsson, Peter (författare)
- KTH,Bioteknologi
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- Uhlén, Mathias (författare)
- KTH,Bioteknologi
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- Hober, Sophia (författare)
- KTH,Bioteknologi
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(creator_code:org_t)
- Elsevier BV, 2001
- 2001
- Engelska.
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 22:1, s. 120-127
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- Escherichia coli (E. coli) single-stranded binding protein (SSB) is a valuable protein for various biotechnical applications, such as PCR and DNA sequencing, Here we describe an efficient expression and purification scheme where the tendency of SSB to aggregate at low salt concentration and high protein concentration is avoided. The method contains fewer steps of purification and results in high protein yield, compared to previous published protocols. In our protocol, cells are harvested after cultivation overnight and SSB is isolated by ammonium sulfate precipitation followed by anion-exchange chromatography. The yield from a 2-liter fed-batch fermenter is 2 g protein, which is higher than all production methods for SSB earlier reported, Moreover, the two classical isolation steps combined in the purification scheme are robust, cost-efficient, and suitable for scaling up. The resulting SSB is pure and a correctly folded tetramer with an apparent binding to single-stranded DNA with a K-D of 10(-8) M, as determined by surface plasmon resonance.
Nyckelord
- SSB
- expression
- purification
- E. coli
- detected magnetic-resonance
- acid unwinding protein
- ssb gene-product
- deoxyribonucleic-acid
- tryptophan residues
- amplification
- purification
- complexes
- mutagenesis
- sequences
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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