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1.
LA Sommer MA Meier SA Dames 《Protein science : a publication of the Protein Society》2012,21(10):1566-1570
The expression of peptides and proteins as fusions to the B1 domain of streptococcal protein G (GB1) is very popular since GB1 often improves the solubility of the target protein and because the first purification step using IgG affinity chromatography is simple and efficient. However, the following protease digest is not always complete or can result in a digest of the target protein. In addition, a further purification step such as RP-HPLC has to be used to get rid of the GB1 tag and undigested fusion protein. Because the protease digest and the following purification step are not only time-consuming but generally also expensive, we tested if GB1 fusion proteins can directly be used for NMR interaction studies using lipids or membrane-mimetics. Based on NMR binding studies using only the GB1 part, this fusion tag does not significantly interact with different membrane-mimetics such as micelles, bicelles, or liposomes. Thus spectral changes observed using GB1-fusion proteins indicate lipid- and membrane interactions of the target protein. The method was initially established to probe membrane interactions of a large number of mutants of the FATC domain of the ser/thr kinase TOR. To demonstrate the usefulness of the approach, we show NMR binding data for the wild type protein and a leucine to alanine mutant. 相似文献
2.
Hammarström M Woestenenk EA Hellgren N Härd T Berglund H 《Journal of structural and functional genomics》2006,7(1):1-14
We have studied the effect of solubilising N-terminal fusion proteins on the yield of target protein after removal of the fusion partner and subsequent purification using immobilised metal ion affinity chromatography. We compared the yield of 45 human proteins produced from four different expression vectors: three having an N-terminal solubilising fusion protein (the GB1-domain, thioredoxin, or glutathione S-transferase) followed by a protease cleavage site and a His tag, and one vector having only an N-terminal His tag. We have previously observed a positive effect on solubility for proteins produced as fusion proteins compared to proteins produced with only a His tag in Escherichia coli. We find this effect to be less pronounced when we compare the yields of purified target protein after removal of the solubilising fusion although large target-dependent variations are seen. On average, the GB1+His fusion gives significantly higher final yields of protein than the thioredoxin+His fusion or the His tag, whereas GST+His gives lower yields. We also note a strong correlation between solubility and target protein size, and a correlation between solubility and the presence of peptide fragments that are predicted to be natively disordered.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. 相似文献
3.
Tomohide Saio Kenji Ogura Masashi Yokochi Yoshihiro Kobashigawa Fuyuhiko Inagaki 《Journal of biomolecular NMR》2009,44(3):157-166
Paramagnetic lanthanide ions fixed in a protein frame induce several paramagnetic effects such as pseudo-contact shifts and
residual dipolar couplings. These effects provide long-range distance and angular information for proteins and, therefore,
are valuable in protein structural analysis. However, until recently this approach had been restricted to metal-binding proteins,
but now it has become applicable to non-metalloproteins through the use of a lanthanide-binding tag. Here we report a lanthanide-binding
peptide tag anchored via two points to the target proteins. Compared to conventional single-point attached tags, the two-point
linked tag provides two to threefold stronger anisotropic effects. Though there is slight residual mobility of the lanthanide-binding
tag, the present tag provides a higher anisotropic paramagnetic effect.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
4.
Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR. 总被引:9,自引:4,他引:5
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J. R. Huth C. A. Bewley B. M. Jackson A. G. Hinnebusch G. M. Clore A. M. Gronenborn 《Protein science : a publication of the Protein Society》1997,6(11):2359-2364
Two protein expression vectors have been designed for the preparation of NMR samples. The vectors encode the immunoglobulin-binding domain of streptococcal protein G (GB1 domain) linked to the N-terminus of the desired proteins. This fusion strategy takes advantage of the small size, stable fold, and high bacterial expression capability of the GB1 domain to allow direct NMR spectroscopic analysis of the fusion protein by 1H-15N correlation spectroscopy. Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the solubility and stability of a protein can be determined. In addition, 15N-labeling of peptides and their testing for DNA binding are facilitated. Several examples are presented that demonstrate the usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand-receptor interactions, using 15N-labeled GB1-peptide fusions and unlabeled target. 相似文献
5.
Amy M. Ruschak Justine D. Rose Michael P. Coughlin Tomasz L. Religa 《Protein science : a publication of the Protein Society》2013,22(11):1646-1654
The low solubility of many proteins hinders large scale expression and purification as well as biophysical measurements. Here, we devised a general strategy to solubilize a protein by conjugating it at a solvent‐exposed position to a 6 kDa protein that was re‐engineered to be highly soluble. We applied this method to the CARD domain of Apoptosis‐associated speck‐like protein containing a CARD (ASC), which represents one member of a class of proteins that are notoriously prone to aggregation. Attachment of the tag to a cysteine residue, introduced by site‐directed mutagenesis at its self‐association interface, improved the solubility of the ASC CARD over 50‐fold under physiological conditions. Although it is not possible to use nuclear magnetic resonance (NMR) to obtain a high quality 2D correlation spectrum of the wild type domain under physiological conditions, we demonstrate that NMR relaxation parameters of the solubilized variant are sufficiently improved to facilitate virtually any demanding measurement. The method shown here represents a straightforward approach for dramatically increasing protein solubility, enabled by ease of labeling as well as flexibility in tag placement with minimal perturbation to the target. © 2013 The Protein Society 相似文献
6.
Xingfu Xu Peter H. J. Keizers Wolfgang Reinle Frank Hannemann Rita Bernhardt Marcellus Ubbink 《Journal of biomolecular NMR》2009,43(4):247-254
Yeast cytochrome c and bovine adrenodoxin form a dynamic electron transfer complex, which is a pure encounter complex. It is demonstrated that
the dynamic nature of the interaction can readily be probed by using a rigid lanthanide tag attached to cytochrome c. The tag, Caged Lanthanide NMR Probe 5, induces pseudocontact shifts and residual dipolar couplings and does not perturb the
binding interface. Due to the dynamics in the complex, residual dipolar couplings in adrenodoxin are very small. Simulation
shows that cytochrome c needs to sample a large part of the surface of adrenodoxin to explain the small degree of alignment observed for adrenodoxin.
The applied method provides a simple and straightforward way to observe dynamics in protein complexes or domain–domain mobility
without the need for external alignment media.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
7.
The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology 总被引:1,自引:0,他引:1
Sofia J. Costa André Almeida António Castro Lucília Domingues Hüseyin Besir 《Applied microbiology and biotechnology》2013,97(15):6779-6791
The Escherichia coli host system is an advantageous choice for simple and inexpensive recombinant protein production but it still presents bottlenecks at expressing soluble proteins from other organisms. Several efforts have been taken to overcome E. coli limitations, including the use of fusion partners that improve protein expression and solubility. New fusion technologies are emerging to complement the traditional solutions. This work evaluates two novel fusion partners, the Fh8 tag (8 kDa) and the H tag (1 kDa), as solubility enhancing tags in E. coli and their comparison to commonly used fusion partners. A broad range comparison was conducted in a small-scale screening and subsequently scaled-up. Six difficult-to-express target proteins (RVS167, SPO14, YPK1, YPK2, Frutalin and CP12) were fused to eight fusion tags (His, Trx, GST, MBP, NusA, SUMO, H and Fh8). The resulting protein expression and solubility levels were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after protein purification and after tag removal. The Fh8 partner improved protein expression and solubility as the well-known Trx, NusA or MBP fusion partners. The H partner did not function as a solubility tag. Cleaved proteins from Fh8 fusions were soluble and obtained in similar or higher amounts than proteins from the cleavage of other partners as Trx, NusA or MBP. The Fh8 fusion tag therefore acts as an effective solubility enhancer, and its low molecular weight potentially gives it an advantage over larger solubility tags by offering a more reliable assessment of the target protein solubility when expressed as a fusion protein. 相似文献
8.
The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production
of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified
cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The
pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the
soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein
G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show
that a C-terminal proline tag, which is invisible in 1H-15N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were
amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized
to improve the expression and purification of various proteins for NMR analysis. 相似文献
9.
We describe a generic protocol for the overproduction and purification of recombinant proteins in Escherichia coli. The strategy utilizes a dual His6-maltose binding protein (HisMBP) affinity tag that can be removed from the target protein by digestion of the fusion protein at a designed site by tobacco etch virus protease. The MBP moiety serves to enhance the solubility and promote the proper folding of its fusion partners, and the polyhistidine tag facilitates its purification to homogeneity. This protocol is divided into three stages, each of which takes approximately 1 week to complete: (i) construction of a HisMBP fusion vector; (ii) a pilot experiment to assess the yield and solubility of the target protein; and (iii) the large-scale production and purification of the target protein. 相似文献
10.
The necessity to acquire large multidimensional datasets, a basis for assignment of NMR resonances, results in long data acquisition
times during which substantial degradation of a protein sample might occur. Here we propose a method applicable for such a
protein for automatic assignment of backbone resonances by direct inspection of multidimensional NMR spectra. In order to
establish an optimal balance between completeness of resonance assignment and losses of cross-peaks due to dynamic processes/degradation
of protein, assignment of backbone resonances is set as a stirring criterion for dynamically controlled targeted nonlinear
NMR data acquisition. The result is demonstrated with the 12 kDa 13C,15 N-labeled apo-form of heme chaperone protein CcmE, where hydrolytic cleavage of 29 C-terminal amino acids is detected. For
this protein, 90 and 98% of manually assignable resonances are automatically assigned within 10 and 40 h of nonlinear sampling
of five 3D NMR spectra, respectively, instead of 600 h needed to complete the full time domain grid. In addition, resonances
stemming from degradation products are identified. This study indicates that automatic resonance assignment might serve as
a guiding criterion for optimal run-time allocation of NMR resources in applications to proteins prone to degradation.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
11.
Philippe S. Nadaud Jonathan J. Helmus Christopher P. Jaroniec 《Biomolecular NMR assignments》2007,1(1):117-120
Complete 13C and 15N assignments of the B3 IgG-binding domain of protein G (GB3) in the microcrystalline solid phase, obtained using 2D and 3D
MAS NMR, are presented. The chemical shifts are used to predict the protein backbone conformation and compared with solution-state
shifts.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
12.
In proteomics research, generation of recombinant proteins in their native, soluble form with large quantity is often a challenging task. To tackle the expression difficulties, different expression vectors with distinct affinity fusion tags, i.e. pET-43.1a (N-utilization substance A tag), pMAL-cRI (maltose binding protein tag) (MBP tag), pGEX-4T-2 (glutathione S-transferase tag), and pET-15b (hexahistidine tag) were compared for their effects on the productivity and solubility, which were assessed by SDS-PAGE and immunoblotting, of the integrin betaA domain. The incubation temperatures were tested for its effects on these parameters. Our data suggested that MBP tag enhanced the yield and solubility of the betaA domain protein, which can also be recognized using an anti-CD18 antibody, at room temperature incubation. Thus, the nature of fusion partner chosen for expression in bacteria and its incubation temperature would significantly affect the yield and solubility of the recombinant target protein. 相似文献
13.
Sumo is one of the fusion tags commonly used to enhance the expression and the solubility of recombinant proteins. One advantage of using sumo is that the removal of the sumo tag is highly specific because its recognition by a sumo protease is determined by its structural characteristics, instead of the sequence of a short peptide. Recently, it was reported that sumo could also be used as a protease recognition site to facilitate the removal of other fusion tags, such as MBP, when sumo itself is not suitable to enhance the solubility of a particular target protein. Using sumo as a recognition site is highly desirable when the target protein needs to have its native N terminus. However, constructing such a plasmid involves more than one cloning step because the N terminus of the target protein needs to be the next residue after the diglycine of sumo. Here, we report the construction of a new vector with a mutant sumo tag. The incorporation of a Pvu II site near the 3′ end of tag coding sequence enables quick construction of plasmids for producing proteins with native termini. Its usage includes producing recombinant food allergens for studying conformational IgE epitopes. 相似文献
14.
15.
Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7. 相似文献
16.
Shinji Tanaka Yasushi Kawata Gottfried Otting Nicholas E. Dixon Katsumi Matsuzaki Masaru Hoshino 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(4):866-871
A novel chaperonin-encapsulation system for NMR measurements has been designed. The single-ring variant SR398 with an ATPase deficient mutation of GroEL, also known as chaperonin, bound co-chaperonin GroES irreversibly, forming a stable cage to encapsulate a target protein. A small GroEL-binding tag made it possible to perform all steps of the encapsulation under near physiological conditions while retaining the native conformation of the target protein. About half of the SR398/GroES cages encapsulated target protein molecules. As binding only depends on the 12-residue tag sequence, this encapsulation method is applicable to a large number of proteins. Isolation of the target proteins in the molecular cage of chaperonin will allow the study of highly aggregation-prone proteins by solution NMR. 相似文献
17.
Lindsay J. Sperling Andrew J. Nieuwkoop Andrew S. Lipton Deborah A. Berthold Chad M. Rienstra 《Journal of biomolecular NMR》2010,46(2):149-155
Magic-angle spinning (MAS) solid-state NMR (SSNMR) spectroscopy of uniformly-13C,15N labeled protein samples provides insight into atomic-resolution chemistry and structure. Data collection efficiency has
advanced remarkably in the last decade; however, the study of larger proteins is still challenged by relatively low resolution
in comparison to solution NMR. In this study, we present a systematic analysis of SSNMR protein spectra acquired at 11.7,
17.6 and 21.1 Tesla (1H frequencies of 500, 750, and 900 MHz). For two protein systems—GB1, a 6 kDa nanocrystalline protein and DsbA, a 21 kDa nanocrystalline
protein—line narrowing is demonstrated in all spectral regions with increasing field. Resolution enhancement is greatest in
the aliphatic region, including methine, methylene and methyl sites. The resolution for GB1 increases markedly as a function
of field, and for DsbA, resolution in the C–C region increases by 42%, according to the number of peaks that can be uniquely
picked and integrated in the 900 MHz spectra when compared to the 500 MHz spectra. Additionally, chemical exchange is uniquely
observed in the highest field spectra for at least two isoleucine Cδ1 sites in DsbA. These results further illustrate the
benefits of high-field MAS SSNMR spectroscopy for protein structural studies. 相似文献
18.
His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors 总被引:2,自引:0,他引:2
Woestenenk EA Hammarström M van den Berg S Härd T Berglund H 《Journal of structural and functional genomics》2004,5(3):217-229
We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at a total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative affect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins. 相似文献
19.
Highly efficient expression and purification system of small-size protein domains in Escherichia coli for biochemical characterization 总被引:1,自引:0,他引:1
Bao WJ Gao YG Chang YG Zhang TY Lin XJ Yan XZ Hu HY 《Protein expression and purification》2006,47(2):599-606
It is often essential to focus the study on the small-size domains of large proteins in eukaryotic cells in the post-genomic era, but the low expression level, insolubility, and instability of the domains have been continuing to hinder the massive purification of domain peptides for structural and biological investigation. In this work, a highly efficient expression and purification system based on a small-size fusion partner GB1 and histidine tag was utilized to solve these problems. Two vectors, namely pGBTNH and pGBH, were constructed to improve expression and facilitate purification. The linker and thrombin cleavage site have been optimized for minimal degradation during purification process. This system has been tested for eight domain peptides varying in size, linker, hydrophobicity, and predicted secondary structure. The results indicate that this system is achievable to produce these domain peptides with high solubility and stability for further biochemical characterization. Moreover, the fusion protein without the linker and thrombin cleavage site is also suitable for spectroscopic studies especially for NMR structural elucidation, if the target peptide is prone to precipitation or easily degraded during purification. This system will be beneficial to the research field of structure and function of small domain and peptide fragment. 相似文献
20.
Lei Shi Nathaniel J. Traaseth Raffaello Verardi Alessandro Cembran Jiali Gao Gianluigi Veglia 《Journal of biomolecular NMR》2009,44(4):195-205
To fully describe the fold space and ultimately the biological function of membrane proteins, it is necessary to determine
the specific interactions of the protein with the membrane. This property of membrane proteins that we refer to as structural topology cannot be resolved using X-ray crystallography or solution NMR alone. In this article, we incorporate into XPLOR-NIH a hybrid
objective function for membrane protein structure determination that utilizes solution and solid-state NMR restraints, simultaneously
defining structure, topology, and depth of insertion. Distance and angular restraints obtained from solution NMR of membrane
proteins solubilized in detergent micelles are combined with backbone orientational restraints (chemical shift anisotropy
and dipolar couplings) derived from solid-state NMR in aligned lipid bilayers. In addition, a supplementary knowledge-based
potential, E
z (insertion depth potential), is used to ensure the correct positioning of secondary structural elements with respect to a
virtual membrane. The hybrid objective function is minimized using a simulated annealing protocol implemented into XPLOR-NIH software
for general use.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献