The role of changing loop conformations in streptavidin versions engineered for high-affinity binding of the Strep-tag II peptide

The role of changing loop conformations in streptavidin versions engineered for high-affinity binding of the Strep-tag II peptide
The affinity system based mostly on the substitute peptide ligand Strep-tag® II and engineered tetrameric streptavidin, often known as Strep-Tactin®, gives enticing functions for the research of recombinant proteins, from detection and purification to useful immobilization.
To additional enhance binding of the Strep-tag II to streptavidin we have now subjected two protruding loops that form its ligand pocket for the peptide – as a substitute of D-biotin acknowledged by the pure protein – to iterative random mutagenesis.
Sequence analyses of hits from useful screening assays revealed a number of surprising structural motifs, comparable to a disulfide bridge on the base of 1 loop, substitute of the essential residue Trp120 by Gly and a two-residue deletion within the second loop. The mutant m1-9 (dubbed Strep-Tactin XT) confirmed strongly enhanced affinity in the direction of the Strep-tag II, which was additional boosted in case of the bivalent Twin-Strep-tag®.
4 consultant streptavidin mutants had been crystallized in complicated with the Strep-tag II and their X-ray constructions had been solved at excessive resolutions. As well as, the crystal construction of the complicated between Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent mode of binding and explaining the experimentally noticed avidity impact.
Our research illustrates the structural plasticity of streptavidin as a scaffold for ligand binding and divulges interplay modes that might have been tough to foretell. As end result, Strep-Tactin XT gives a handy reagent for the kinetically secure immobilization of recombinant proteins fused with the Twin-Strep-tag. The opportunity of reversibly dissociating such complexes merely with D-biotin allows useful research in protein science in addition to cell biology.

Streptag II fusion know-how for the modification and immobilization of lipase B from Candida antarctica (CALB).

Fusion tags – amino acid sequences which can be genetically coded to be expressed as connected moieties to a protein – have the potential to boost the exercise of native enzyme, allow particular purification of the enzyme, and promote easy and environment friendly immobilization of enzymes onto materials helps. On this work, we exhibit the impact of a Strep-tag II fusion tag on the properties of free and immobilized lipase B from Candida antarctica (CALB).
The gene encoding the mature portion of CALB was codon-optimized and cloned in pASG-IBA2 plasmid for expression in E. coli. Purified recombinant Strep-tag II CALB was immobilized to Strep-Tactin based mostly help by way of affinity binding, and the immobilized and free Strep-tag II CALB had been in comparison with a business CALB. Following modification, the enzyme could possibly be selectively purified from tradition media with no observable non-specific binding.
The catalytic effectivity of the purified fusion-tagged enzyme was considerably larger than that of the business CALB in its free kind. Immobilization of the fusion-tagged enzyme to Strep-Tactin modified crosslinked agarose help yielded a catalytically energetic enzyme; nevertheless, the okaycat of the immobilized enzyme was considerably lowered in comparison with the free tagged enzyme.
This work signifies {that a} C-terminus Strep-tag II fusion tag could also be employed to enhance the catalytic effectivity of free CALB, however might not be appropriate for immobilized functions that make use of binding of the enzyme to a Strep-Tactin-modified help.
After injecting their genome into the bacterial host cell, bacteriophages have to convert the host metabolism towards environment friendly phage manufacturing. For this, particular proteins have developed which work together with key host proteins to inhibit, activate or redirect the perform of those proteins.
Since 70% of the presently annotated phage genes are hypothetical proteins of unknown perform, the identification and characterization of those phage proteins concerned in host-phage protein-protein interactions stays difficult.
Right here, we describe a way to determine phage proteins concerned in host-phage protein-protein interactions utilizing a mix of affinity purifications and mass spectrometry analyses. A bacterial pressure is engineered through which a bacterial goal protein is fused to a Strep-tag® II on the C-terminal finish. This pressure is contaminated with a selected bacteriophage, adopted by an affinity purification of the tagged protein which permits the copurification of all bacterial and phage particular interacting proteins.
After SDS-PAGE evaluation and an in-gel trypsin digestion, the purified interacting proteins are recognized by mass spectrometry evaluation. The identification of phage proteins concerned in interactions gives first hints towards the elucidation of the organic perform of those proteins.

Inclusion of Streptag II in design of antigen receptors for T-cell immunotherapy.

Adoptive immunotherapy with genetically engineered T cells has the potential to deal with most cancers and different illnesses. The introduction of Strep-tag II sequences into particular websites in artificial chimeric antigen receptors or pure T-cell receptors of numerous specificities gives engineered T cells with a marker for identification and fast purification, a way for tailoring spacer size of chimeric receptors for optimum perform, and a useful aspect for selective antibody-coated, microbead-driven, large-scale growth.
These receptor designs facilitate cGMP manufacturing of pure populations of engineered T cells for adoptive T-cell therapies and allow in vivo monitoring and retrieval of transferred cells for downstream analysis functions.

Streptag II Mutant Maltose-binding Protein for Reagentless Fluorescence Sensing.

Maltose-binding protein (MBP) is a periplasmic binding protein present in Gram unfavorable micro organism. MBP is concerned in maltose transport and bacterial chemotaxis; it binds to maltose and maltodextrins comprising α(1-4)-glucosidically linked linear glucose polymers and α(1-4)-glucosidically linked cyclodextrins. Upon ligand binding, MBP adjustments its conformation from an open to a closed kind.
This molecular recognition-transducing a ligand-binding occasion right into a bodily one-renders MBP a perfect candidate for biosensor growth. Right here, we describe the development of a Strep-tag II mutant MBP for reagentless fluorescence sensing. malE, which encodes MBP, was amplified.

 The role of changing loop conformations in streptavidin versions engineered for high-affinity binding of the Strep-tag II peptide

A cysteine residue was launched by site-directed mutagenesis to make sure a single label attachment at a selected web site with a thiol-specific fluorescent probe. An environmentally delicate fluorophore (IANBD amide) was covalently connected to the launched thiol group and analysed by fluorescence sensing. The tagged mutant MBP (D95C) was purified (molecular measurement, ∼42 kDa).

Strep-Tag II Antibody

EUR 370

Strep-Tag II Antibody

EUR 146

Strep-Tag II Blocking Peptide

EUR 153

Mouse anti Strep II-Tag mAb

AE066 100 ul
EUR 256

Strep II tag Mouse Monoclonal Antibody

T603-100ul 100ul
EUR 252

Strep II tag Mouse Monoclonal Antibody

T603-50ul 50ul
EUR 187

Strep II tag Mouse Monoclonal Antibody

T0017 100 ug
EUR 438

Strep-tag Antibody

  • EUR 356.00
  • EUR 537.00
  • EUR 217.00
  • 100 ul
  • 200 ul
  • 30 ul

Strep-Tag Antibody

  • EUR 328.00
  • EUR 272.00
  • 100 ug
  • 50 ug

Strep tag antibody

70R-12234 100 ug
EUR 494
Description: Rabbit polyclonal Strep tag antibody

Strep-Tag Monoclonal Antibody

EM1015-100ul 100ul
EUR 279
Description: A Mouse Monoclonal antibody against Strep-Tag. This antibody is tested and validated for WB, ELISA
The fluorescence measurements of the IANBD-labelled Strep-tag II-D95C within the answer section confirmed an considerable change in fluorescence depth (dissociation fixed, 7.6±1.75 μM). Our mutant MBP retains maltose-binding exercise and is appropriate for reagentless fluorescence sensing.

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