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  • 1.
    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 protein2006Ingår i: Journal of Structural and Functional Genomics, ISSN 1345-711X, E-ISSN 1570-0267, Vol. 7, nr 1, s. 1-14Artikel i tidskrift (Refereegranskat)
    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.

  • 2.
    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 genomics2003Ingår i: Analytical biochemistry, ISSN 0003-2697, Vol. 318, nr 1, s. 71-79Artikel i tidskrift (Refereegranskat)
    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.

  • 3.
    Woestenenk, Esmeralda A.
    KTH, Tidigare Institutioner, Bioteknologi.
    Protein production, characterization and structure determination in structural genomics2004Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    This thesis covers the process from expression of a heterologous gene in Escherichia coli to structure determination of a protein by nuclear magnetic resonance (NMR) spectroscopy.

    The first part concerns structural genomics-related parallel screening studies on the effect of fusion tags (in particular the His tag) on protein solubility and the use of fusion tags in fast, parallel purification protocols intended for initial biophysical characterization of human proteins produced in E. coli. It was found that for most proteins the His tag has a negative influence on protein solubility. This influence appears to be more pronounced for our C-terminal His tag than for the N-terminal His tags used in this study. Moreover, high ratios of soluble per total protein do not always guarantee a high yield of soluble protein after purification, as different vector - target protein combinations result in large differences in host cell growth rates. Protein purification protocols for different fusion tags were developed that make it possible to express, purify and study structural properties of low concentration samples of 15N-labeled proteins in one or two days.

    The second part of this thesis describes the assignment and solution structure determination of ribosomal protein L18 of Thermus thermophilus. The protein is a mixed α/β structure with two α-helices on one side of a four-stranded β-sheet. Comparison to RNA-bound L18 showed that the protein to a large extent adopts identical structures in free and bound states, with exception of the loop regions and the flexible N-terminus.

    Keywords: protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination, ribosomal protein

  • 4.
    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 thermophilus2000Ingår i: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 17, nr 3, s. 273-274Artikel i tidskrift (Refereegranskat)
  • 5.
    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 fold2002Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 363, nr 3, s. 553-561Artikel i tidskrift (Refereegranskat)
    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.

  • 6.
    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 vectors2004Ingår i: Journal of Structural and Functional Genomics, ISSN 1345-711X, E-ISSN 1570-0267, Vol. 5, nr 3, s. 217-229Artikel i tidskrift (Refereegranskat)
    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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