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  • 1.
    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.

  • 2.
    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.

  • 3.
    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)
  • 4.
    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.

  • 5.
    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)
  • 6.
    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.

  • 7.
    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.

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