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Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
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2006 (English)In: Journal of Structural and Functional Genomics, ISSN 1345-711X, E-ISSN 1570-0267, Vol. 7, no 1, 1-14 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2006. Vol. 7, no 1, 1-14 p.
Keyword [en]
Escherichia coli; Fusion protein; High throughput; His tag; Solubility; Structural genomics
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-8996DOI: 10.1007/s10969-005-9003-7Scopus ID: 2-s2.0-33747676822OAI: oai:DiVA.org:kth-8996DiVA: diva2:14522
Note
QC 20100826Available from: 2006-01-16 Created: 2006-01-16 Last updated: 2010-11-30Bibliographically approved
In thesis
1. 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
2. Probing the prospects for structural genomics: expression and characterization of human disease-related proteins in E. coli
Open this publication in new window or tab >>Probing the prospects for structural genomics: expression and characterization of human disease-related proteins in E. coli
2005 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

Today a lot of research is directed towards the development of methods to make protein structure determination faster and more generalised. Automated, general methods for protein expression and purification, robotics for crystallisation and automatic methods for data collection will take structural biology into a new era, that of structural genomics. In two previously performed studies applications were developed for potential use in a structural genomics context. In the first, a fast method to screen for soluble proteins expressed in E. coli using seven N-terminal fusion proteins as solubility enhancing tags was developed. The method was tested on 32 small human proteins suitable for structural study with NMR (Hammarström et al, 2002). In the second, a biophysical screen for fast identification of proteins suitable for structure determination was developed (Woestenenk et al, 2003).

This thesis is an extension of the two previous studies. We have tested the solubility enhancing effect of four N-terminal fusion proteins on 47 cancer and developmental disease related human target proteins, of sizes ranging from 6.6 to 126.5 kDa. We have indications that the solubility enhancing effect is dependent on target protein size. Small proteins of size less than 20 kDa are more likely to be generally affected by the solubility enhancing effect of the fusion partners.

All targets that were overexpressed in the present screen and ten previously uncharacterized targets, related to cancer, from an earlier project were selected for biophysical characterization trials. We were able to purify 15 targets for NMR, CD spectroscopy or both methods. A majority of the targets were estimated to be unstructured or partially unstructured. Only one target was considered directly suitable for structure determination using NMR. The solubility screen and biophysical results were compared to DisEMBL™ and GlobPlot™ disorder predictions of all targets. We found a close match between biophysical data and prediction results. We concluded that, even though we obtain an increased solubility using fusion proteins, many of our targets are likely to be unstructured or partially unstructured and are thus not suitable for structure determination.

Place, publisher, year, edition, pages
Stockholm: KTH, 2005. v, 66 p.
Keyword
Biochemistry, Biokemi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-289 (URN)91-7283-946-5 (ISBN)
Supervisors
Note
QC 20101130Available from: 2005-07-11 Created: 2005-07-11 Last updated: 2010-11-30Bibliographically approved

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Hammarström, MartinWoestenenk, EsmeraldaHellgren, NiklasHärd, TorleifBerglund, Helena
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