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His tag effect on solubility of human proteins produced in Escherichia coli: A comparison between four expression vectors
KTH, Tidigare Institutioner, Bioteknologi.
KTH, Tidigare Institutioner, Bioteknologi.
KTH, Tidigare Institutioner, Bioteknologi.
KTH, Tidigare Institutioner, Bioteknologi.
Visa övriga samt affilieringar
2004 (Engelska)Ingår i: Journal of Structural and Functional Genomics, ISSN 1345-711X, E-ISSN 1570-0267, Vol. 5, nr 3, s. 217-229Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2004. Vol. 5, nr 3, s. 217-229
Nyckelord [en]
Affinity purification, High throughput, His tag, Protein purification, Solubility, Structural genomics
Nationell ämneskategori
Biologiska vetenskaper
Identifikatorer
URN: urn:nbn:se:kth:diva-5329DOI: 10.1023/B:jsfg.0000031965.37625.0ePubMedID: 15503425Scopus ID: 2-s2.0-3543050105OAI: oai:DiVA.org:kth-5329DiVA, id: diva2:8630
Tillgänglig från: 2004-10-01 Skapad: 2004-10-01 Senast uppdaterad: 2017-12-01Bibliografiskt granskad
Ingår i avhandling
1. Protein production, characterization and structure determination in structural genomics
Öppna denna publikation i ny flik eller fönster >>Protein production, characterization and structure determination in structural genomics
2004 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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

Ort, förlag, år, upplaga, sidor
Bioteknologi, 2004
Nyckelord
Biology, protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination, Biologi
Nationell ämneskategori
Biologiska vetenskaper
Identifikatorer
urn:nbn:se:kth:diva-29 (URN)91-7283-838-8 (ISBN)
Disputation
2004-10-01, FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Handledare
Tillgänglig från: 2004-10-01 Skapad: 2004-10-01 Senast uppdaterad: 2012-03-22
2. 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

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Woestenenk, Esmeralda A.Hammarström, Martinvan den Berg, SusanneHärd, TorleifBerglund, Helena
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