Optimisation of a multivalent Strep tag for protein detection

Stadler, Lukas Kurt Josef; Ferrigno, Paul Ko; Davis, Jason J.

doi:10.1016/j.bpc.2010.09.005

Cited by 8 publications

(5 citation statements)

References 44 publications

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“…In addition to tags previously employed [ 13 , 18 ], these vectors include ones, such as tandem Strep -tag II sequences and MBP preceded by a signal sequence, that are compatible with attachment to the extracytoplasmic termini of membrane proteins. The tighter binding of Streptactin to the tandem Strep -tag II than the single Strep -tag II [ 19 ] allows purification of membrane proteins to near homogeneity in a single step.…”

Section: Introductionmentioning

confidence: 99%

A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters

Hao

Huysmans

et al. 2015

PLoS ONE

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Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.

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Section: Introductionmentioning

confidence: 99%

A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters

Hao

Huysmans

et al. 2015

PLoS ONE

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“…Because the biotin binding pockets in NTV and AV have similar surface structures, one may expect that NTV – like AV – is unable to bind ST. It has been reported that the binding affinity of ST to STV can be further increased to nanomolar levels when using multiple tandem STs [27]. It is also shown that in protein purification, having multiple tandem STs improves the binding affinity to STN [24].…”

Section: Introductionmentioning

confidence: 99%

“…To increase the stability [24], [27], our method uses a tandem two STs (tST)-STN linkage to couple two molecules A and B, where both A and B can potentially be either DNA or protein of arbitrary size. Here we demonstrate the coupling of Maltose Binding Protein to a 920 nm long dsDNA.…”

Section: Introductionmentioning

confidence: 99%

A Polypeptide-DNA Hybrid with Selective Linking Capability Applied to Single Molecule Nano-Mechanical Measurements Using Optical Tweezers

2013

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Many applications in biosensing, biomaterial engineering and single molecule biophysics require multiple non-covalent linkages between DNA, protein molecules, and surfaces that are specific yet strong. Here, we present a novel method to join proteins and dsDNA molecule at their ends, in an efficient, rapid and specific manner, based on the recently developed linkage between the protein StrepTactin (STN) and the peptide StrepTag II (ST). We introduce a two-step approach, in which we first construct a hybrid between DNA and a tandem of two STs peptides (tST). In a second step, this hybrid is linked to polystyrene bead surfaces and Maltose Binding Protein (MBP) using STN. Furthermore, we show the STN-tST linkage is more stable against forces applied by optical tweezers than the commonly used biotin-Streptavidin (STV) linkage. It can be used in conjunction with Neutravidin (NTV)-biotin linkages to form DNA tethers that can sustain applied forces above 65 pN for tens of minutes in a quarter of the cases. The method is general and can be applied to construct other surface-DNA and protein-DNA hybrids. The reversibility, high mechanical stability and specificity provided by this linking procedure make it highly suitable for single molecule mechanical studies, as well as biosensing and lab on chip applications.

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“…We have shown that engineered nonantibody probe molecules such as peptide aptamers based on the thioredoxin scaffold or Affimers based on the Stefin A scaffold , can be used to replace antibodies in many detection platforms. Of key relevance here is recent work showing that Affimers can be used for optical (surface plasmon resonance or SPR) and electrochemical detection of various proteins. − Affimers differ from immunoglobulins in being monomeric (which would be useful in establishing assays for the monomeric form of CRP, where antibodies that recognize a repeating epitope in a pentamer would be predicted to have weaker affinity for a monovalent target) and from other nonantibody probe molecules in being able to present three independent surfaces for interaction that mimic the large diversity and surface area used by antibodies. , In addition, Affimer libraries have been made for screening in yeast two hybrid and phage display formats, extending their versatility …”

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confidence: 99%

Sensitive Affimer and Antibody Based Impedimetric Label-Free Assays for C-Reactive Protein

et al. 2012

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Optimisation of a multivalent Strep tag for protein detection

Cited by 8 publications

References 44 publications

A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters

A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters

A Polypeptide-DNA Hybrid with Selective Linking Capability Applied to Single Molecule Nano-Mechanical Measurements Using Optical Tweezers

Sensitive Affimer and Antibody Based Impedimetric Label-Free Assays for C-Reactive Protein

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