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

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.     

Applications of a peptide ligand for streptavidin: the Strep-tag

The Strep-tag constitutes a nine amino acid-peptide that binds specifically to streptavidin and occupies the same pocket where biotin is normally complexed. Since the Strep-tag participates in a reversible interaction it can be applied for the efficient purification of corresponding fusion proteins on affinity columns with immobilized streptavidin. Elution of the bound recombinant protein can be effected under mild buffer conditions by competition with biotin or a suitable derivative. In addition, Strep-tag fusion proteins can be easily detected in immunochemical assays, like Western blots or ELISAs, by means of commercially available streptavidin-enzyme conjugates. The Strep-tag/streptavidin system has been systematically optimized over the past years, including the engineering of streptavidin itself. Structural insight into the molecular mimicry between the peptide and biotin was furthermore gained from X-ray crystallographic analysis. As a result the system provides a reliable and versatile tool in recombinant protein chemistry. Exemplary applications of the Strep-tag are discussed in this review.     

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

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners.     

An improved protein bioreactor: efficient product isolation during in vitro protein biosynthesis via affinity tag

In vitro protein biosynthesis became a powerful technology for biochemical research. Beside the determination of structure and function in vitro selection of proteins is also of great interest. In most cases the use of a synthesized protein for further applications depends on its purity. For this purpose the in vitro production and purification of proteins with short affinity tails was established. A cell-free protein synthesis system was employed to produce bovine heart fatty acid-binding protein and bacterial chloramphenicol acetyltransferase with and without fusion of the Strep-tag affinity peptide. The quantitative removal of fusion protein during cell-free synthesis from a batch reaction and a semicontinuous flow cell-free reactor were achieved. No significant influence of the Strep-tag and the conditions during the affinity chromatography on maturation or activity of the proteins were observed. The product removal from the continuous flow cell-free reactor is still an only partially solved problem, because the use of ultrafiltration membranes has some limitations. The results document that it should be possible to avoid these limitations by introducing an affinity system.     

The periplasmic E. coli chaperone Skp is a trimer in solution: biophysical and preliminary crystallographic characterization

The 'seventeen kilodalton protein' Skp confers transient solubility on outer membrane proteins during biogenesis in Gram-negative bacteria. Here we report a first biophysical characterization of this chaperone itself, which also possesses biotechnological potential in the production of recombinant proteins. Using cross-linking and gel filtration methods, we found that Skp forms a stable homo-trimer in solution. Following thermal denaturation, monitored by CD spectroscopy, this chaperone refolds with high efficiency but exhibits a pronounced hysteresis between the un- and refolding transitions. Using the recombinant protein equipped with the Strep-tag II at its N-terminus, suitable crystallization conditions for Skp were found. A first data set was collected to 2.60 A resolution.     

Strep-tag II and Twin-Strep Based Cassettes for Protein Tagging by Homologous Recombination and Characterization of Endogenous Macromolecular Assemblies in Saccharomyces cerevisiae

Peptide sequences fused to a gene of interest facilitate the isolation of proteins or protein complexes from cell extracts. In the case of fluorescent protein tags, the tagged protein can be visually localized in living cells. To tag endogenous genes, PCR-based homologous recombination is a powerful approach used in the yeast Saccharomyces cerevisiae. This approach uses short, homologous DNA sequences that flank the tagging cassette to direct recombination. Here, we constructed a set of plasmids, whose sequences were optimized for codon usage in yeast, for Strep-tag II and Twin-Strep tagging in S. cerevisiae. Some plasmids also contain sequences encoding for a fluorescent protein followed by the purification tag. We demonstrate using the yeast pyruvate dehydrogenase (PDH) complex that these plasmids can be used to purify large protein complexes efficiently. We furthermore demonstrate that purification from the endogenous pool using the Strep-tag system results in functionally active complexes. Finally, using the fluorescent tags, we show that a kinase and a phosphatase involved in regulating the activity of the PDH complex localize in the cells’ mitochondria. In conclusion, our cassettes can be used as tools for biochemical, functional, and structural analyses of endogenous multi-protein assemblies in yeast.     

A disulfide bond-containing alkaline phosphatase triggers a BdbC-dependent secretion stress response in Bacillus subtilis

The gram-positive bacterium Bacillus subtilis secretes high levels of proteins into its environment. Most of these secretory proteins are exported from the cytoplasm in an unfolded state and have to fold efficiently after membrane translocation. As previously shown for alpha-amylases of Bacillus species, inefficient posttranslocational protein folding is potentially detrimental and stressful. In B. subtilis, this so-called secretion stress is sensed and combated by the CssRS two-component system. Two known members of the CssRS regulon are the htrA and htrB genes, encoding potential extracytoplasmic chaperone proteases for protein quality control. In the present study, we investigated whether high-level production of a secretory protein with two disulfide bonds, PhoA of Escherichia coli, induces secretion stress in B. subtilis. Our results show that E. coli PhoA production triggers a relatively moderate CssRS-dependent secretion stress response in B. subtilis. The intensity of this response is significantly increased in the absence of BdbC, which is a major determinant for posttranslocational folding of disulfide bond-containing proteins in B. subtilis. Our findings show that BdbC is required to limit the PhoA-induced secretion stress. This conclusion focuses interest on the BdbC-dependent folding pathway for biotechnological production of proteins with disulfide bonds in B. subtilis and related bacilli.     

链霉亲和素及其亲和系统的蛋白质进化

链霉亲和素/生物素（Streptavidin/Biotin）体系作为目前已知的最高亲和力作用体系,已在生物学研究中获得广泛应用。本文针对Streptavidin/Biotin和Strep-Tactin/Strep-tag两个相关系统的演化,分别从链霉亲和素蛋白的结构改造、亲和肽标签优化等方面进行了较为详细的归纳。通过对链霉亲和素蛋白各种突变体的优缺点的比较,有助于实际应用中选择合适的Streptavidin突变体。本文通过对链霉亲和素蛋白质进化的综述,可帮助更准确地理解市场上各种链霉亲和素蛋白的功能和用途,并为深入研究链霉亲和素蛋白的进化提供参考。     

The AviD-tag, a NeutrAvidin/avidin specific peptide affinity tag for the immobilization and purification of recombinant proteins

The widespread success of affinity tags throughout the biological sciences has prompted interest in developing new and convenient labeling strategies. Affinity tags are well-established tools for recombinant protein immobilization and purification. More recently these tags have been utilized for selective biological targeting towards multiplexed protein detection in numerous imaging applications as well as for drug-delivery. Recently, we discovered a phage-display selected cyclic peptide motif that was shown to bind selectively to NeutrAvidin and avidin but not to the structurally similar streptavidin. Here, we have exploited this selectivity to develop an affinity tag based on the evolved DRATPY moiety that is orthogonal to known Strep-tag technologies. As proof of principle, the divalent AviD-tag (Avidin-Di-tag) was expressed as a Green Fluorescent Protein variant conjugate and exhibited superior immobilization and elution characteristics to the first generation Strep-tag and a monovalent DRATPY GFP-fusion protein analogue. Additionally, we demonstrate the potential for a peptide based orthogonal labeling strategy involving our divalent AviD-tag in concert with existing streptavidin-based affinity reagents. We believe the AviD-tag and its unique recognition properties will provide researchers with a useful new affinity reagent and tool for a variety of applications in the biological and chemical sciences.     

Complementary impact of paralogous Oxa1-like proteins of Bacillus subtilis on post-translocational stages in protein secretion

In mitochondria, chloroplasts, and Gram-negative eubacteria, Oxa1p(-like) proteins are critical for the biogenesis of membrane proteins. Here we show that the Gram-positive eubacterium Bacillus subtilis contains two functional Oxa1p orthologues, denoted SpoIIIJ and YqjG. The presence of either SpoIIIJ or YqjG is required for cell viability. Whereas SpoIIIJ is required for sporulation, YqjG is dispensable for this developmental process. The stability of two membrane proteins was found to be mildly affected upon SpoIIIJ limitation in the absence of YqjG. Surprisingly, the topology and stability of other membrane proteins remained unaffected under these conditions. In contrast, SpoIIIJ- and YqjG-limiting conditions resulted in a strong post-translocational defect in the stability of secretory proteins. Together, these data indicate that SpoIIIJ and YqjG of B. subtilis are involved in both membrane protein biogenesis and protein secretion. However, the reduced stability of secretory proteins seems to be the most prominent phenotype of SpoIIIJ/YqjG-depleted B. subtilis cells. In conclusion, our observations show that SpoIIIJ and YqjG have different, but overlapping functions in B. subtilis. Most importantly, it seems that different members of the Oxa1p protein family have acquired at least partly distinct, species-specific, functions that are essential for life.     

Functional implementation of the posttranslational SecB-SecA protein-targeting pathway in Bacillus subtilis

Bacillus subtilis and its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria, B. subtilis does not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein-targeting pathway into B. subtilis by coexpressing SecB from Escherichia coli together with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of the B. subtilis SecA were replaced by the corresponding part of SecA from E. coli. In vitro pulldown experiments showed that, in contrast to B. subtilis SecA, the hybrid SecA protein gained the ability to efficiently bind to E. coli SecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that the secretion of both proteins by B. subtilis was significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished the secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.     

TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway

The recent discovery of a ubiquitous translocation pathway, specifically required for proteins with a twin-arginine motif in their signal peptide, has focused interest on its membrane-bound components, one of which is known as TatC. Unlike most organisms of which the genome has been sequenced completely, the Gram-positive eubacterium Bacillus subtilis contains two tatC-like genes denoted tatCd and tatCy. The corresponding TatCd and TatCy proteins have the potential to be involved in the translocation of 27 proteins with putative twin-arginine signal peptides of which approximately 6-14 are likely to be secreted into the growth medium. Using a proteomic approach, we show that PhoD of B. subtilis, a phosphodiesterase belonging to a novel protein family of which all known members are synthesized with typical twin-arginine signal peptides, is secreted via the twin-arginine translocation pathway. Strikingly, TatCd is of major importance for the secretion of PhoD, whereas TatCy is not required for this process. Thus, TatC appears to be a specificity determinant for protein secretion via the Tat pathway. Based on our observations, we hypothesize that the TatC-determined pathway specificity is based on specific interactions between TatC-like proteins and other pathway components, such as TatA, of which three paralogues are present in B. subtilis.     

Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase.

We have cloned the operon coding for the Bacillus subtilis S complex, which has been proposed to be a component in protein secretion machinery. A lambda gt10 library of B. subtilis was screened with antiserum directed against the Staphylococcus aureus membrane-bound ribosome protein complex, which is homologous to the B. subtilis S complex. Two positive overlapping lambda clones were sequenced. The S-complex operon, 5 kilobases in size, was shown to contain four open reading frames and three putative promoters, which are located upstream of the first, the third, and the last gene. The four proteins encoded by the operon are 42, 36, 48, and 50 kilodaltons in size. All of these proteins were recognized by antisera separately raised against each protein of the S. aureus membrane-bound ribosome protein and B. subtilis S complexes, thus verifying the S-complex identity of the lambda clones. Sequence analysis revealed that all four proteins of the B. subtilis S complex are homologous to the four subunits of the human pyruvate dehydrogenase (PDH). Also, the N terminus of the 48-kilodalton protein was found to have 70% amino acid identity with the N-terminal 211 amino acids, determined so far, from the E2 subunit of B. stearothermophilus PDH. Furthermore, chromosomal mapping of the S-complex operon gave a linkage to a marker gene located close to the previously mapped B. subtilis PDH genes. Thus, the S complex is evidently identical to the B. subtilis PDH, which has been shown to contain four subunits with molecular weights very similar to those of the S complex. Therefore, we propose that the S complex is not a primary component of protein secretion.     

pAM401-based shuttle vectors that enable overexpression of promoterless genes and one-step purification of tag fusion proteins directly from Enterococcus faecalis

Two novel Enterococcus faecalis-Escherichia coli shuttle vectors that utilize the promoter and ribosome binding site of bacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli and E. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless beta-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. beta-Galactosidase was expressed in E. coli and E. faecalis at levels of 10(3) and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.