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Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3)
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0002-7067-9173
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.
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).

Place, publisher, year, edition, pages
2010. Vol. 69, no 2, 159-167 p.
Keyword [en]
E. coli, BL21(DE3), Rosetta(DE3), Expression strain, Protein, production, High-throughput, rare codon clusters, expression, translation, purification, genes, bias
URN: urn:nbn:se:kth:diva-19042DOI: 10.1016/j.pep.2009.08.017ISI: 000272653300006ScopusID: 2-s2.0-70449525585OAI: diva2:337089
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2013-11-20Bibliographically approved
In thesis
1. Proteome wide protein production
Open this publication in new window or tab >>Proteome wide protein production
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.
Trita-BIO-Report, ISSN 1654-2312 ; 2013:17
protein production, Escherichia coli, transcription, promoter, translation, rare codon, high-throughput, screening
National Category
Natural Sciences
urn:nbn:se:kth:diva-134215 (URN)978-91-7501-913-0 (ISBN)
Public defence
2013-12-06, FR4, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (English)

QC 20131120

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

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