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Probing the prospects for structural genomics: expression and characterization of human disease-related proteins in E. coli
KTH, School of Biotechnology (BIO).
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 [en]
Biochemistry
Keyword [sv]
Biokemi
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-289ISBN: 91-7283-946-5 (print)OAI: oai:DiVA.org:kth-289DiVA: diva2:8619
Supervisors
Note
QC 20101130Available from: 2005-07-11 Created: 2005-07-11 Last updated: 2010-11-30Bibliographically approved
List of papers
1. Rapid screening for improved solubility of small human proteins produced as fusio proteins in Escherichia coli.
Open this publication in new window or tab >>Rapid screening for improved solubility of small human proteins produced as fusio proteins in Escherichia coli.
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2002 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 11, no 2, 313-321 p.Article in journal (Refereed) Published
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.

Keyword
Fusion proteins, solubility, recombination, genetic disorder, structural genomics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-8994 (URN)10.1110/ps.22102 (DOI)000173352700014 ()
Note
QC 20100825Available from: 2006-01-16 Created: 2006-01-16 Last updated: 2017-12-14Bibliographically approved
2. Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein
Open this publication in new window or tab >>Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein
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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.

Keyword
Escherichia coli; Fusion protein; High throughput; His tag; Solubility; Structural genomics
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8996 (URN)10.1007/s10969-005-9003-7 (DOI)2-s2.0-33747676822 (Scopus ID)
Note
QC 20100826Available from: 2006-01-16 Created: 2006-01-16 Last updated: 2017-12-14Bibliographically approved

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