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Protein production, characterization and structure determination in structural genomics
KTH, Superseded Departments, Biotechnology.
2004 (English)Doctoral thesis, comprehensive summary (Other scientific)
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

Place, publisher, year, edition, pages
Bioteknologi , 2004.
Keyword [en]
Biology, protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination
Keyword [sv]
Biologi
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-29ISBN: 91-7283-838-8 (print)OAI: oai:DiVA.org:kth-29DiVA: diva2:8634
Public defence
2004-10-01, FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Available from: 2004-10-01 Created: 2004-10-01 Last updated: 2012-03-22
List of papers
1. His tag effect on solubility of human proteins produced in Escherichia coli: A comparison between four expression vectors
Open this publication in new window or tab >>His tag effect on solubility of human proteins produced in Escherichia coli: A comparison between four expression vectors
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2004 (English)In: Journal of Structural and Functional Genomics, ISSN 1345-711X, E-ISSN 1570-0267, Vol. 5, no 3, 217-229 p.Article in journal (Refereed) Published
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.

Keyword
Affinity purification, High throughput, His tag, Protein purification, Solubility, Structural genomics
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-5329 (URN)10.1023/B:jsfg.0000031965.37625.0e (DOI)15503425 (PubMedID)2-s2.0-3543050105 (Scopus ID)
Available from: 2004-10-01 Created: 2004-10-01 Last updated: 2017-12-01Bibliographically approved
2. Screening methods to determine biophysical properties of proteins in structural genomics
Open this publication in new window or tab >>Screening methods to determine biophysical properties of proteins in structural genomics
2003 (English)In: Analytical biochemistry, ISSN 0003-2697, Vol. 318, no 1, 71-79 p.Article in journal (Refereed) Published
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.

Keyword
CD spectroscopy; Heterologous gene expression; High throughput; NMR spectroscopy; Protein purification; Structural genomics
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-5330 (URN)10.1016/S0003-2697(03)00162-3 (DOI)000183495900010 ()12782033 (PubMedID)
Note
QC 20100826Available from: 2004-10-01 Created: 2004-10-01 Last updated: 2010-12-07Bibliographically approved
3. Assignment and secondary structure identification of the ribosomal protein L18 from Thermus thermophilus
Open this publication in new window or tab >>Assignment and secondary structure identification of the ribosomal protein L18 from Thermus thermophilus
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2000 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 17, no 3, 273-274 p.Article in journal (Refereed) Published
Keyword
resonance assignment, ribosomal protein, RNA-binding protein, Thermus thermophilus
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-38463 (URN)10.1023/A:1008319622724 (DOI)000088223000011 ()
Note
LetterAvailable from: 2011-08-26 Created: 2011-08-26 Last updated: 2017-12-08Bibliographically approved
4. The solution structure of ribosomal protein L18 from Thermus thermophilus reveals a conserved RNA-binding fold
Open this publication in new window or tab >>The solution structure of ribosomal protein L18 from Thermus thermophilus reveals a conserved RNA-binding fold
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2002 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 363, no 3, 553-561 p.Article in journal (Refereed) Published
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.

Keyword
NMR spectroscopy, Protein structure, Ribosome, RNA-binding protein
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-5332 (URN)10.1042/0264-6021:3630553 (DOI)000175651600015 ()11964156 (PubMedID)
Available from: 2004-10-01 Created: 2004-10-01 Last updated: 2017-12-01Bibliographically approved

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