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Rapid screening for improved solubility of small human proteins produced as fusio proteins in Escherichia coli.
KTH, Tidigare Institutioner                               , Bioteknologi.
KTH, Tidigare Institutioner                               , Bioteknologi.
KTH, Tidigare Institutioner                               , Bioteknologi.
KTH, Tidigare Institutioner                               , Bioteknologi.
Visa övriga samt affilieringar
2002 (Engelska)Ingår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 11, nr 2, s. 313-321Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2002. Vol. 11, nr 2, s. 313-321
Nyckelord [en]
Fusion proteins, solubility, recombination, genetic disorder, structural genomics
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:kth:diva-8994DOI: 10.1110/ps.22102ISI: 000173352700014OAI: oai:DiVA.org:kth-8994DiVA, id: diva2:14520
Anmärkning
QC 20100825Tillgänglig från: 2006-01-16 Skapad: 2006-01-16 Senast uppdaterad: 2017-12-14Bibliografiskt granskad
Ingår i avhandling
1. Protein production and purification in structural genomics
Öppna denna publikation i ny flik eller fönster >>Protein production and purification in structural genomics
2006 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 2006. s. vii, 67
Nyckelord
Recombinational cloning, Escherichia coli, gene expression, fusion proteins, solubility, circular dichroism, nuclear magnetic resonance
Nationell ämneskategori
Annan industriell bioteknik
Identifikatorer
urn:nbn:se:kth:diva-589 (URN)91-7178-239-7 (ISBN)
Disputation
2006-01-27, FR4, AlbaNova Huvudbyggnaden, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Handledare
Anmärkning
QC 20100826Tillgänglig från: 2006-01-16 Skapad: 2006-01-16 Senast uppdaterad: 2011-12-08Bibliografiskt granskad
2. Probing the prospects for structural genomics: expression and characterization of human disease-related proteins in E. coli
Öppna denna publikation i ny flik eller fönster >>Probing the prospects for structural genomics: expression and characterization of human disease-related proteins in E. coli
2005 (Engelska)Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 2005. s. v, 66
Nyckelord
Biochemistry, Biokemi
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-289 (URN)91-7283-946-5 (ISBN)
Handledare
Anmärkning
QC 20101130Tillgänglig från: 2005-07-11 Skapad: 2005-07-11 Senast uppdaterad: 2010-11-30Bibliografiskt granskad

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Förlagets fulltexthttp://dx.doi.org/10.1110/ps.22102

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Hammarström, MartinHellgren, Niklasvan den Berg, SusanneBerglund, HelenaHärd, Torleif
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