Endre søk
Begrens søket
1 - 13 of 13
RefereraExporteraLink til resultatlisten
Permanent link
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Treff pr side
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sortering
  • Standard (Relevans)
  • Forfatter A-Ø
  • Forfatter Ø-A
  • Tittel A-Ø
  • Tittel Ø-A
  • Type publikasjon A-Ø
  • Type publikasjon Ø-A
  • Eldste først
  • Nyeste først
  • Skapad (Eldste først)
  • Skapad (Nyeste først)
  • Senast uppdaterad (Eldste først)
  • Senast uppdaterad (Nyeste først)
  • Disputationsdatum (tidligste først)
  • Disputationsdatum (siste først)
  • Standard (Relevans)
  • Forfatter A-Ø
  • Forfatter Ø-A
  • Tittel A-Ø
  • Tittel Ø-A
  • Type publikasjon A-Ø
  • Type publikasjon Ø-A
  • Eldste først
  • Nyeste først
  • Skapad (Eldste først)
  • Skapad (Nyeste først)
  • Senast uppdaterad (Eldste først)
  • Senast uppdaterad (Nyeste først)
  • Disputationsdatum (tidligste først)
  • Disputationsdatum (siste først)
Merk
Maxantalet träffar du kan exportera från sökgränssnittet är 250. Vid större uttag använd dig av utsökningar.
  • 1.
    Dincbas-Renqvist, Vildan
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Lendel, Christofer
    KTH, Skolan för bioteknologi (BIO).
    Dogan, Jakob
    KTH, Tidigare Institutioner, Bioteknologi.
    Wahlberg, Elisabet
    KTH, Tidigare Institutioner, Bioteknologi.
    Härd, Torleif
    Göteborgs Universitet.
    Thermodynamics of folding, stabilization, and binding in an engineered protein-protein complex2004Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, nr 36, s. 11220-11230Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We analyzed the thermodynamics of a complex protein-protein binding interaction using the (engineered) Z(SPA-1) affibody and it's Z domain binding partner as a model. Free Z(SPA-1) exists in an equilibrium between a molten-globule-like (MG) state and a completely unfolded state, wheras a well-ordered structure is observed in the Z:Z(SPA-1) complex. The thermodynamics of the MG state unfolding equilibrium can be separated from the thermodynamics of binding and stabilization by combined analysis of isothermal titration calorimetry data and a separate van't Hoff analysis of thermal unfolding. We find that (i) the unfolding equilibrium of free Z(SPA-1) has only a small influence on effective binding affinity, that (ii) the Z:Z(SPA-1) interface is inconspicuous and structure-based energetics calculations suggest that it should be capable of supporting strong binding, but that (iii) the conformational stabilization of the MG state to a well-ordered structure in the Z:Z(SPA-1) complex is associated with a large change in conformational entropy that opposes binding.

  • 2.
    Dogan, Jakob
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Lendel, Christofer
    KTH, Skolan för bioteknologi (BIO).
    Härd, Torleif
    Göteborgs Universitet.
    NMR assignments of the free and bound-state protein components of an anti-idiotypic affibody complex2006Inngår i: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 36, s. (Electronic publication ahead of print Feb. 6; doi:10.1007/s10858-005-5350-8)Artikkel i tidsskrift (Fagfellevurdert)
  • 3.
    Hammarström, Martin
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hellgren, Niklas
    KTH, Tidigare Institutioner                               , Bioteknologi.
    van den Berg, Susanne
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Berglund, Helena
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Härd, Torleif
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Rapid screening for improved solubility of small human proteins produced as fusio proteins in Escherichia coli.2002Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 11, nr 2, s. 313-321Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subclone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N-terminal fusion proteins and an N-terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR-sized and nonmembrane human proteins as soluble fusion proteins in E. coli.

  • 4.
    Hammarström, Martin
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Woestenenk, Esmeralda
    KTH, Skolan för bioteknologi (BIO).
    Hellgren, Niklas
    KTH, Skolan för bioteknologi (BIO).
    Härd, Torleif
    Berglund, Helena
    KTH, Skolan för bioteknologi (BIO).
    Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein2006Inngår i: Journal of Structural and Functional Genomics, ISSN 1345-711X, E-ISSN 1570-0267, Vol. 7, nr 1, s. 1-14Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 5.
    Lendel, Christofer
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Dincbas-Renqvist, Vildan
    KTH, Tidigare Institutioner, Bioteknologi.
    Flores, Alexander
    KTH, Tidigare Institutioner, Bioteknologi.
    Wahlberg, Elisabet
    KTH, Tidigare Institutioner, Bioteknologi.
    Dogan, Jakob
    KTH, Tidigare Institutioner, Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner, Bioteknologi.
    Härd, Torleif
    Biophysical characterization of ZSPA-1-A phage-display selected binder to protein A2004Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 13, nr 8, s. 2078-2088Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Affibodies are a novel class of binding proteins selected from phagemid libraries of the Z domain from staphylococcal protein A. The Z(SPA-1) affibody was selected as a binder to protein A, and it binds the parental Z domain with micromolar affinity. In earlier work we determined the structure of the Z:Z(SPA-1) complex and noted that Z(SPA-1) in the free state exhibits several properties characteristic of a molten globule. Here we present a more detailed biophysical investigation of Z(SPA-1) and four Z(SPA-1) mutants with the objective to understand these properties. The characterization includes thermal and chemical denaturation profiles, ANS binding assays, size exclusion chromatography, isothermal titration calorimetry, and an investigation of structure and dynamics by NMR. The NMR characterization of Z(SPA-1) was facilitated by the finding that trimethylamine N-oxide (TMAO) stabilizes the molten globule conformation in favor of the fully unfolded state. All data taken together lead us to conclude the following: (1) The topology of the molten globule conformation of free Z(SPA-1) is similar to that of the fully folded structure in the Z-bound state; (2) the extensive mutations in helices 1 and 2 destabilize these without affecting the intrinsic stability of helix 3; (3) stabilization and reduced aggregation can be achieved by replacing mutated residues in Z(SPA-1) with the corresponding wild-type Z residues. This stabilization is better correlated to changes in helix propensity than to an expected increase in polar versus nonpolar surface area of the fully folded state.

  • 6.
    Lendel, Christofer
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Dogan, Jakob
    KTH, Skolan för bioteknologi (BIO).
    Härd, Torleif
    Göteborgs Universitet.
    Structural basis for molecular recognition in an affibody:affibody complex2006Inngår i: Asia-Pacific Journal of Molecular Biology and Biotechnology, ISSN 0128-7451, Vol. 359, nr 5, s. 1293-1304Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Affibody molecules constitute a class of engineered binding proteins based on the 58-residue three-helix bundle Z domain derived from staphylococcal protein A (SPA). Affibody proteins are selected as binders to target proteins by phage display of combinatorial libraries in which typically 13 side-chains on the surface of helices 1 and 2 in the Z domain have been randomized. The Z(Taq):anti-Z(Taq) affibody-affibody complex, consisting of Z(Taq), originally selected as a binder to Taq DNA polymerase, and anti-Z(Taq), selected as binder to Z(Taq), is formed with a dissociation constant K-d similar to 100 nM. We have determined high-precision solution structures of free Z(Taq) and anti-Z(Taq), and the Z(Taq):anti-Z(Taq) complex under identical experimental conditions (25 degrees C in 50 mM NaCl with 20 mM potassium phosphate buffer at pH 6.4). The complex is formed with helices 1 and 2 of anti-Z(Taq) in perpendicular contact with helices 1 and 2 of Z(Taq). The interaction surface is large (similar to 1670 angstrom(2)) and unusually non-polar (70 %) compared to other protein-protein complexes. It involves all varied residues on anti-Z(Taq), most corresponding (Taq DNA polymerase binding) side-chains on Z(Taq), and several additional side-chain and backbone contacts. Other notable features include a substantial rearrangement (induced fit) of aromatic side-chains in Z(Taq) upon binding, a close contact between glycine residues in the two subunits that might involve aliphatic glycine H alpha to backbone carbonyl hydrogen bonds, and four hydrogen bonds made by the two guanidinium (NH2)-H-eta groups of an arginine side-chain. Comparisons of the present structure with other data for affibody proteins and the Z domain suggest that intrinsic binding properties of the originating SPA surface might be inherited by the affibody binders. A thermodynamic characterization of Z(Taq) and anti-Z(Taq) is presented in an accompanying paper.

  • 7.
    Lendel, Christofer
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Wahlberg, Elisabet
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Berglund, Helena
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Eklund, Malin
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Härd, Torleif
    KTH, Tidigare Institutioner                               , Bioteknologi.
    1H, 13C and 15N resonance assignments of an affibody-target complex2002Inngår i: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 24, nr 3, s. 271-272Artikkel i tidsskrift (Fagfellevurdert)
  • 8.
    Wahlberg, Elisabet
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Härd, Torleif
    Conformational Stabilization of an Engineered Binding Protein2006Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 128, nr 23, s. 7651-7660Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We analyzed the thermodynamic basis for improvement of a binding protein by disulfide engineering. The Z(SPA-1) affibody binds to its Z domain binding partner with a dissociation constant K-d = 1.6 mu M, and previous analyses suggested that the moderate \affinity is due to the conformational heterogeneity of free Z(SPA-1) rather than to a suboptimal binding interface. Studies of five stabilized Z(SPA-1) double cystein mutants show that it is possible to improve the affinity by an order of magnitude to K-d = 130 nM, which is close to the range (20 to 70 nM) observed with natural Z domain binders, without altering the protein-protein interface obtained by phage display. Analysis of the binding thermodynamics reveals a balance between conformational entropy and desolvation entropy: the expected and favorable reduction of conformational entropy in the best-binding Z(SPA-1) mutant is completely compensated by an unfavorable loss of desolvation entropy. This is consistent with a restriction of possible conformations in the disulfide-containing mutant and a reduction of average water-exposed nonpolar surface area in the free state, resulting in a smaller conformational entropy penalty, but also a smaller change in surface area, for binding of mutant compared to wild-type Z(SPA-1). Instead, higher Z domain binding affinity in a group of eight Z(SPA-1) variants correlates with more favorable binding enthalpy and enthalpy- entropy compensation. These results suggest that protein-protein binding affinity can be improved by stabilizing conformations in which enthalpic effects can be fully explored.

  • 9.
    Wahlberg, Elisabet
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Lendel, Christofer
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Helgstrand, Magnus
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Allard, Peter
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Dincbas-Renqvist, Vildan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hedqvist, Anders
    Berglund, Helena
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Härd, Torleif
    KTH, Tidigare Institutioner                               , Bioteknologi.
    An affibody in complex with a target protein: Structure and coupled folding.2003Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 100, nr 6, s. 3185-3190Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Combinatorial protein engineering provides powerful means for functional selection of novel binding proteins. One class of engineered binding proteins, denoted affibodies, is based on the three-helix scaffold of the Z domain derived from staphylococcal protein A. The Z(SPA-1) affibody has been selected from a phage-displayed library as a binder to protein A. Z(SPA-1) also binds with micromolar affinity to its own ancestor, the Z domain. We have characterized the Z(SPA-1) affibody in its uncomplexed state and determined the solution structure of a Z:Z(SPA-1) protein-protein complex. Uncomplexed Z(SPA-1) behaves as an aggregation-prone molten globule, but folding occurs on binding, and the original (Z) three-helix bundle scaffold is fully formed in the complex. The structural basis for selection and strong binding is a large interaction interface with tight steric and polar/nonpolar complementarity that directly involves 10 of 13 mutated amino acid residues on Z(SPA-1). We also note similarities in how the surface of the Z domain responds by induced fit to binding of Z(SPA-1) and Ig Fc, respectively, suggesting that the Z(SPA-1) affibody is capable of mimicking the morphology of the natural binding partner for the Z domain.

  • 10.
    Woestenenk, Esmeeralda A.
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hammarström, Martin
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Härd, Torleif
    Screening methods to determine biophysical properties of proteins in structural genomics2003Inngår i: Analytical biochemistry, ISSN 0003-2697, Vol. 318, nr 1, s. 71-79Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have developed and tested a simple and efficient protein purification method for biophysical screening of proteins and protein fragments by nuclear magnetic resonance (NMR) and optical methods, such as circular dichroism spectroscopy. The method constitutes an extension of previously described protocols for gene expression and protein solubility screening [M. Hammarström et al., (2002), Protein Science 11, 313]. Using the present purification scheme it is possible to take several target proteins, produced as fusion proteins, from cell pellet to NMR spectrum and obtain a judgment on the suitability for further structural or biophysical studies in less than 1 day. The method is independent of individual protein properties as long as the target protein can be produced in soluble form with a fusion partner. Identical procedures for cell culturing, lysis, affinity chromatography, protease cleavage, and NMR sample preparation then initially require only optimization for different fusion partner and protease combinations. The purification method can be automated, scaled up or down, and extended to a traditional purification scheme. We have tested the method on several small human proteins produced in Escherichia coli and find that the method allows for detection of structured proteins and unfolded or molten globule-like proteins.

  • 11.
    Woestenenk, Esmeralda A.
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Allard, Peter
    KTH, Tidigare Institutioner, Bioteknologi.
    Gongadze, G. M.
    Moskalenko, S. E.
    Shcherbakov, D. V.
    Rak, A. V.
    Garber, M B
    Härd, Torleif
    KTH, Tidigare Institutioner, Bioteknologi.
    Berglund, Helena
    KTH, Tidigare Institutioner, Bioteknologi.
    Assignment and secondary structure identification of the ribosomal protein L18 from Thermus thermophilus2000Inngår i: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 17, nr 3, s. 273-274Artikkel i tidsskrift (Fagfellevurdert)
  • 12.
    Woestenenk, Esmeralda A.
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Gongadze, George M.
    Shcherbakov, Dmitry V.
    Rak, Alexey V.
    Garber, Maria B.
    Härd, Torleif
    KTH, Tidigare Institutioner, Bioteknologi.
    Berglund, Helena
    KTH, Tidigare Institutioner, Bioteknologi.
    The solution structure of ribosomal protein L18 from Thermus thermophilus reveals a conserved RNA-binding fold2002Inngår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 363, nr 3, s. 553-561Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have determined the solution structure of ribosomal protein L18 from Thermus thermophilus. L18 is a 12.5 kDa protein of the large subunit of the ribosome and binds to both 5 S and 23 S rRNA. In the uncomplexed state L18 folds to a mixed α/β globular structure with a long disordered N-terminal region. We compared our high-resolution structure with RNA-complexed L 18 from Haloarcula marismortui and T. thermophilus to examine RNA-induced as well as species-dependent structural differences. We also identified T. thermophilus S11 as a structural homologue and found that the structures of the RNA-recognition sites are conserved. Important features, for instance a bulge in the RNA-contacting β-sheet, are conserved in both proteins. We suggest that the L18 fold recognizes a specific RNA motif and that the resulting RNA-protein-recognition module is tolerant to variations in sequence.

  • 13.
    Woestenenk, Esmeralda A.
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Hammarström, Martin
    KTH, Tidigare Institutioner, Bioteknologi.
    van den Berg, Susanne
    KTH, Tidigare Institutioner, Bioteknologi.
    Härd, Torleif
    KTH, Tidigare Institutioner, Bioteknologi.
    Berglund, Helena
    KTH, Tidigare Institutioner, Bioteknologi.
    His tag effect on solubility of human proteins produced in Escherichia coli: A comparison between four expression vectors2004Inngår i: Journal of Structural and Functional Genomics, ISSN 1345-711X, E-ISSN 1570-0267, Vol. 5, nr 3, s. 217-229Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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 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 effect 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 a 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.

1 - 13 of 13
RefereraExporteraLink til resultatlisten
Permanent link
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf