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Protein production and purification in structural genomics
KTH, School of Biotechnology (BIO).
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 [en]
Recombinational cloning, Escherichia coli, gene expression, fusion proteins, solubility, circular dichroism, nuclear magnetic resonance
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-589ISBN: 91-7178-239-7 (print)OAI: oai:DiVA.org:kth-589DiVA: diva2:14524
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
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, 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: 2010-11-30Bibliographically approved
2. 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
3. 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: 2010-11-30Bibliographically approved
4. 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

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