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Screening methods to determine biophysical properties of proteins in structural genomics
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
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.

Place, publisher, year, edition, pages
2003. Vol. 318, no 1, 71-79 p.
Keyword [en]
CD spectroscopy; Heterologous gene expression; High throughput; NMR spectroscopy; Protein purification; Structural genomics
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-5330DOI: 10.1016/S0003-2697(03)00162-3ISI: 000183495900010PubMedID: 12782033OAI: oai:DiVA.org:kth-5330DiVA: diva2:8631
Note
QC 20100826Available from: 2004-10-01 Created: 2004-10-01 Last updated: 2010-12-07Bibliographically approved
In thesis
1. Protein production, characterization and structure determination in structural genomics
Open this publication in new window or tab >>Protein production, characterization and structure determination in structural genomics
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
Biology, protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination, Biologi
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-29 (URN)91-7283-838-8 (ISBN)
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
2. Protein production and purification in structural genomics
Open this publication in new window or tab >>Protein production and purification in structural genomics
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

The number of gene products available for structural and functional study is increasing at an unprecedented rate as a result of the successful whole genome sequencing projects. Systematic structure determination of proteins on a genomic scale, called structural genomics, can significantly contribute to the field of protein science and to functional annotation of newly identified genes.

This thesis covers different aspects of protein production in Eschericiha coli for structural studies in the context of structural genomics. Protocols have been downscaled and standardized to allow for a rapid assessment of the production characteristics for multiple proteins in parallel under a number of different conditions. Foremost, the ability of different proteins and peptide tags to affect the solubility of the recombinant protein when produced as fusion proteins has been systematically studied. Large differences in the success-rate for production of soluble protein in E. coli were found depending on the fusion partner used, with a more than two-fold increase in the number of proteins produced as soluble when comparing the best and the poorest fusion tags. For different constructs with a histidine tag, commonly used to facilitate protein purification, large differences in yield depending on the design of the expression vector were found. When comparing different fusion proteins produced from identical expression vectors, fusions to the GB1 domain were found to result in the highest yield of purified target protein, on average 25 % higher than any of the other fusions.

The suitability for further structural studies was tested at an intermediate scale for proteins that were identified as soluble in the expression screening. For this purpose, protocols for rapid purification and biophysical characterization using nuclear magnetic resonance and circular dichroism spectroscopy were developed and tested on 19 proteins, of which four were structured.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. vii, 67 p.
Keyword
Recombinational cloning, Escherichia coli, gene expression, fusion proteins, solubility, circular dichroism, nuclear magnetic resonance
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-589 (URN)91-7178-239-7 (ISBN)
Public defence
2006-01-27, FR4, AlbaNova Huvudbyggnaden, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100826Available from: 2006-01-16 Created: 2006-01-16 Last updated: 2011-12-08Bibliographically approved

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