http://wolfson.huji.ac.il/purification/PDF/Literature/Arnau2006.pdf

Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins.

The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-tag II fusion proteins--including their complexes with interacting partners--both from bacterial and eukaryotic cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be accomplished within 1 h. A high-affinity monoclonal antibody (StrepMAB-Immo) permits stable immobilization of Strep-tag II fusion proteins to solid surfaces, for example, for surface plasmon resonance analysis. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody (StrepMAB-Classic). Thus, the Strep-tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis.

The Strep -tag system for one-step purification and high-affinity detection or capturing of proteins

A large number of methods have been developed over the years to study protein-protein interactions. Many of these techniques are now available to the nonspecialist researcher thanks to new affordable instruments and/or resource centres. A typical protein-protein interaction study usually starts with an initial screen for novel binding partners. We start this review by describing three techniques that can be used for this purpose: (i) affinity-tagged proteins (ii) the two-hybrid system and (iii) some quantitative proteomic techniques that can be used in combination with, e.g., affinity chromatography and coimmunoprecipitation for screening of protein-protein interactions. We then describe some public protein-protein interaction databases that can be searched to identify previously reported interactions for a given bait protein. Four strategies for validation of protein-protein interactions are presented: confocal microscopy for intracellular colocalization of proteins, coimmunoprecipitation, surface plasmon resonance (SPR) and spectroscopic studies. Throughout the review we focus particularly on the advantages and limitations of each method.

/pdf/methods-for-the-detection-and-analysis-of-protein-protein-47qj8o8wc4.pdf

Methods for the detection and analysis of protein-protein interactions

Summary: The bacterial superantigens are protein toxins that bind to major histocompatibility complex class II and T-cell receptor to stimulate large numbers of T cells. The majority are produced by the Gram-positive organisms Staphylococcus aureus and Streptococcus pyogenes and are the causative agents in toxic shock syndrome, an acute disease caused by the sudden and massive release of T-cell cytokines into the blood stream. The structure and function of the superantigens has revealed a common architecture that is also shared by another group of staphylococcal virulence factors called the superantigen-like proteins (SSL). Together, this family of structurally related molecules highlights how a common pathogenic organism has employed a simple but adaptable protein to generate an armamentarium of potent defense molecules designed to target of the innate and adaptive immune response.

The bacterial superantigen and superantigen-like proteins.

A review on the application of mass spectrometry (MS) to the upper levels of protein organization is presented A description on the processes of generating, transmitting, and analyzing ions formed by protein complexes is presented as well as the experimental conditions theory that are the standards for the investigations In addition, some other approaches are included that can be used in conjunction with MS of intact complexes This review aims to establish MS of intact complexes as part of future structural genomic and proteomic investigations Several other topics discussed include the generation of ions of protein complexes, the transmission and analysis of ions o protein complexes, the activation and dissociation of protein complexes, and lastly, the companion technologies for MS of protein complexes

/pdf/protein-complexes-in-the-gas-phase-technology-for-structural-1lvsurwu0a.pdf

Protein complexes in the gas phase: technology for structural genomics and proteomics.

Identification of protein complexes is the key to understanding cellular functions. In this study, we present a novel method for the identification of multiprotein complexes from mammalian cells. By using the Strep-tag affinity chromatography method, enabling fast and simple one-step purification, coupled with competitive elution under physiological conditions, we successfully purified a PP2A holoenzyme protein complex from a cultured mammalian cancer cell line. We identified, by mass spectrometry, both known and novel interacting proteins for PP2A, and demonstrate that the purified PP2A complex is functional. The benefits and potential applications of the Strep-tag method for protein complex purification are discussed.

Single‐step Strep‐tag® purification for the isolation and identification of protein complexes from mammalian cells

We identified seven novel variants of streptococcal pyrogenic exotoxin G (SPEGG), a superantigen, in Streptococcus dysgalactiae subsp. dysgalactiae or equisimilis isolates from clinical cases of infection in humans and animals. Phylogenetic analysis of the SPEGG variants indicated two clades in the dendrogram: one composed of variants derived from the bacteria isolated from the humans and the other composed of variants from the bacteria isolated from the animals. Bovine peripheral blood mononuclear cells (PBMCs) were stimulated effectively by recombinant SPEGGs (rSPEGGs) expressed in Escherichia coli, while human PBMCs were not stimulated well by any of the rSPEGGs tested. SPEGGs selectively stimulated bovine T cells bearing Vβ1,10 and Vβ4. Bovine serum showed reactivity to the rSPEGG proteins. These results indicated that SPEGGs have properties as superantigens, and it is likely that SPEGGs play a pathogenic role in animals.

https://iai.asm.org/content/iai/75/4/1721.full.pdf

Cloning, Expression, and Characterization of the Superantigen Streptococcal Pyrogenic Exotoxin G from Streptococcus dysgalactiae

Crystal Structure of Streptococcus dysgalactiae-Derived Mitogen Reveals a Zinc-Binding Site and Alterations in TcR Binding

Superantigens are bacterial or viral toxins with potent immunostimulatory properties. Streptococcus dysgalactiae-derived mitogen, a 25 kDa protein, is a recently discovered superantigen isolated from S. dysgalactiae culture supernatant. Sequence considerations suggest that it belongs to a new superantigen family distinct from other superantigens. The protein was expressed in Escherichia coli cells and purified to homogeneity. Crystals were grown at pH 4.2-4.4 in the presence of 18-20%(w/v) PEG 3350 and 0.4 M lithium nitrate. A complete data set to 2.4 A resolution was collected from a single crystal at liquid-nitrogen temperatures using synchrotron radiation. The crystals belong to space group P3/P3(1)/P3(2), with unit-cell parameters a = b = 52.7, c = 62.4 A, gamma = 120 degrees and one molecule in the crystallographic asymmetric unit.

/pdf/expression-purification-and-crystallization-of-streptococcus-475avos07z.pdf

Susanna Saarinen

Papers

Single‐step Strep‐tag® purification for the isolation and identification of protein complexes from mammalian cells

Cloning, Expression, and Characterization of the Superantigen Streptococcal Pyrogenic Exotoxin G from Streptococcus dysgalactiae

Crystal Structure of Streptococcus dysgalactiae-Derived Mitogen Reveals a Zinc-Binding Site and Alterations in TcR Binding

Expression, purification and crystallization of Streptococcus dysgalactiae-derived mitogen.