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Overexpression, rapid isolation, and biochemical characterization of Escherichia coli single-stranded DNA-binding protein
KTH, Tidigare Institutioner                               , Bioteknologi.ORCID-id: 0000-0002-4657-8532
KTH, Tidigare Institutioner                               , Bioteknologi.ORCID-id: 0000-0001-8993-048X
KTH, Tidigare Institutioner                               , Bioteknologi.ORCID-id: 0000-0003-0605-8417
2001 (Engelska)Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 22, nr 1, s. 120-127Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

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.

Ort, förlag, år, upplaga, sidor
2001. Vol. 22, nr 1, s. 120-127
Nyckelord [en]
SSB, expression, purification, E. coli, detected magnetic-resonance, acid unwinding protein, ssb gene-product, deoxyribonucleic-acid, tryptophan residues, amplification, purification, complexes, mutagenesis, sequences
Identifikatorer
URN: urn:nbn:se:kth:diva-20698ISI: 000169169600017OAI: oai:DiVA.org:kth-20698DiVA, id: diva2:339394
Anmärkning
QC 20100525Tillgänglig från: 2010-08-10 Skapad: 2010-08-10 Senast uppdaterad: 2017-12-12Bibliografiskt granskad

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Nilsson, PeterUhlén, MathiasHober, Sophia
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Bioteknologi
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Protein Expression and Purification

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