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Proteome wide protein production
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-7067-9173
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Over a decade after the completion of the human genome, researchers around the world are still wondering what information is hidden in the genome. Although the sequences of all human genes are known, it is still almost impossible to determine much more than the primary protein structure from the coding sequence of a gene. As a result of that, the need for recombinantly produced proteins to study protein structure and function is greater than ever. The main objective of this thesis has been to improve protein production, particularly using Escherichia coli. To improve protein production in Escherichia coli there are a number of different parameters to consider. Two very important parameters in the process of protein production are transcription and translation. To study the influence of differences in transcription rate, target proteins with different characteristics were produced under control of three promoters of different strength (lacUV5, trc and T7). Analyzing the total amount of target protein as well as the amount of soluble protein demonstrated the benefits of using a strong promoter such as T7. However, protein production is also highly dependent on translational efficiency, and a drawback associated with the use of Escherichia coli as host strain is that codons rarely used in this host can have a negative effect on the translation. The influence of using a strain supplied with genes for rare codon tRNAs, such as Rosetta(DE3), instead of the standard host strain BL21(DE3), was therefore evaluated. By using Rosetta(DE3) an improved protein yield for many of the poorly produced proteins was achieved, but more importantly the protein purity was significantly increased for a majority of the proteins. For further understanding of the underlying causes of the positive effects of Rosetta(DE3), the improved purity was thoroughly studied. The cause of this improvement was explained by the fact that Rosetta(DE3) has a significantly better read through of the full sequence during translation and thereby less truncated versions of the full-length protein is formed.  Moreover, the effect of supplementation of rare tRNAs was shown to be highly dependent on the target gene sequence. Surprisingly, it was not the total number of rare codons that determined the benefit of using Rosetta(DE3), instead it was shown that rare arginine codons and to some extent also rare codon clusters had a much bigger impact on the final outcome.

As a result of the increased interest in large-scale studies in the field of proteomics, the need for high-throughput protein production pipelines is greater than ever. For that purpose, a protein production pipeline that allows handling of nearly 300 different proteins per week was set up within the Swedish Human Protein Atlas project. This was achieved by major and minor changes to the original protocol including protein production, purification and analysis. By using this standard setup almost 300 different proteins can be produced weekly, with an overall success rate of 81%. To further improve the success rate it has been shown that by adding an initial screening step, prior high-throughput protein production, unnecessary protein production can be avoided. A plate based micro-scale screening protocol for parallel production and verification of 96 proteins was developed. In that, protein production was performed using the EnBase® cultivation technology followed by purification based on immobilized metal ion affinity chromatography. The protein products were finally verified using matrix-assisted laser desorption ionization time-of-flight MS. By using this method, proteins that will be poorly produced can be sorted out prior high-throughput protein production.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. , xiii, 67 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2013:17
Keyword [en]
protein production, Escherichia coli, transcription, promoter, translation, rare codon, high-throughput, screening
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:kth:diva-134215ISBN: 978-91-7501-913-0 (print)OAI: oai:DiVA.org:kth-134215DiVA: diva2:665536
Public defence
2013-12-06, FR4, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20131120

Available from: 2013-11-20 Created: 2013-11-20 Last updated: 2013-11-20Bibliographically approved
List of papers
1. High-throughput protein production--lessons from scaling up from 10 to 288 recombinant proteins per week
Open this publication in new window or tab >>High-throughput protein production--lessons from scaling up from 10 to 288 recombinant proteins per week
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2009 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 4, no 1, 51-57 p.Article in journal (Refereed) Published
Abstract [en]

The demand for high-throughput recombinant protein production has markedly increased with the increased activity in the field of proteomics. Within the Human Protein Atlas project recombinantly produced human protein fragments are used for antibody production. Here we describe how the protein expression and purification protocol has been optimized in the project to allow for han- dling of nearly 300 different proteins per week. The number of manual handling steps has been significantly reduced (from 18 to 9) and the protein purification has been completely automated.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2009
Keyword
High-throughput, Protein production, Proteomics
National Category
Natural Sciences
Identifiers
urn:nbn:se:kth:diva-74493 (URN)10.1002/biot.200800183 (DOI)19039781 (PubMedID)2-s2.0-64549086069 (Scopus ID)
Note
QC 20120228Available from: 2012-02-28 Created: 2012-02-03 Last updated: 2017-12-08Bibliographically approved
2. Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3)
Open this publication in new window or tab >>Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3)
2010 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 69, no 2, 159-167 p.Article in journal (Refereed) Published
Abstract [en]

The effect of two Escherichia coli expression strains on the production of recombinant human protein fragments was evaluated. High-throughput protein production projects, such as the Swedish Human Protein Atlas project, are dependent on high protein yield and purity. By changing strain from E. coli BL21(DE3) to E. coli Rosetta(DE3) the overall success rate of the protein production has increased dramatically. The Rosetta(DE3) strain compensates for a number of rare codons. Here, we describe how the protein expression of human gene fragments in E. coli strains BL21(DE3) and Rosetta(DE3) was evaluated in two stages. Initially a test set of 68 recombinant proteins that previously had been expressed in BL21(DE3) was retransformed and expressed in Rosetta(DE3). The test set generated very positive results with an improved expression yield and a significantly better purity of the protein product which prompted us to implement the Rosetta(DE3) strain in the high-throughput protein production. Except for analysis of protein yield and purity the sequences were also analyzed regarding number of rare codons and rare codon clusters. The content of rare codons showed to have a significant effect on the protein purity. Based on the results of this study the atlas project permanently changed expression strain to Rosetta(DE3).

Keyword
E. coli, BL21(DE3), Rosetta(DE3), Expression strain, Protein, production, High-throughput, rare codon clusters, expression, translation, purification, genes, bias
Identifiers
urn:nbn:se:kth:diva-19042 (URN)10.1016/j.pep.2009.08.017 (DOI)000272653300006 ()2-s2.0-70449525585 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2013-11-20Bibliographically approved
3. Enhancing the protein production levels in Escherichia coli with a strong promoter
Open this publication in new window or tab >>Enhancing the protein production levels in Escherichia coli with a strong promoter
2011 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 5, 729-739 p.Article in journal (Refereed) Published
Abstract [en]

In biotechnology, the use of Escherichia coli for recombinant protein production has a long tradition, although the optimal production conditions for certain proteins are still not evident. The most favorable conditions for protein production vary with the gene product. Temperature and induction conditions represent parameters that affect total protein production, as well as the amount of soluble protein. Furthermore, the choice of promoter and bacterial strain will have large effects on the production of the target protein. In the present study, the effects of three different promoters (T7, trc and lacUV5) on E. coli production of target proteins with different characteristics are presented. The total amount of target protein as well as the amount of soluble protein were analyzed, demonstrating the benefits of using a strong promoter such as T7. To understand the underlying causes, transcription levels have been correlated with the total amount of target protein and protein solubility in vitro has been correlated with the amount of soluble protein that is produced. In addition, the effects of two different E. coli strains, BL21(DE3) and Rosetta(DE3), on the expression pattern were analyzed. It is concluded that the regulation of protein production is a combination of the transcription and translation efficiencies. Other important parameters include the nucleotide-sequence itself and the solubility of the target protein.

Keyword
Escherichia coli, promoter, protein production, transcription, translation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-31353 (URN)10.1111/j.1742-4658.2010.07991.x (DOI)000287448800004 ()
Note
QC 20110317Available from: 2011-03-17 Created: 2011-03-14 Last updated: 2013-11-20Bibliographically approved
4. Parallel production and verification of protein products using a novel high-throughput screening method
Open this publication in new window or tab >>Parallel production and verification of protein products using a novel high-throughput screening method
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2011 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 6, no 8, 1018-1025 p.Article in journal (Refereed) Published
Abstract [en]

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase (R) cultivation technology was applied, which enables cultivation in as small volumes as 150 mu L. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale.

Keyword
His-tag, IMAC, Micro-scale, Protein production, Protein purification, Screening
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-39519 (URN)10.1002/biot.201000430 (DOI)000294108100012 ()
Funder
Knut and Alice Wallenberg Foundation
Available from: 2011-09-20 Created: 2011-09-12 Last updated: 2017-12-08Bibliographically approved
5. In-depth study of the positive effects of Escherichia coli Rosetta(DE3) on recombinant protein production
Open this publication in new window or tab >>In-depth study of the positive effects of Escherichia coli Rosetta(DE3) on recombinant protein production
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(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-134217 (URN)
Note

QS 2013

Available from: 2013-11-20 Created: 2013-11-20 Last updated: 2013-11-20Bibliographically approved

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