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High-throughput protein production--lessons from scaling up from 10 to 288 recombinant proteins per week
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.
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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. Vol. 4, no 1, 51-57 p.
Keyword [en]
High-throughput, Protein production, Proteomics
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:kth:diva-74493DOI: 10.1002/biot.200800183PubMedID: 19039781Scopus ID: 2-s2.0-64549086069OAI: oai:DiVA.org:kth-74493DiVA: diva2:489710
Note
QC 20120228Available from: 2012-02-28 Created: 2012-02-03 Last updated: 2017-12-08Bibliographically 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.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2013:17
Keyword
protein production, Escherichia coli, transcription, promoter, translation, rare codon, high-throughput, screening
National Category
Natural Sciences
Identifiers
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)
Opponent
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Note

QC 20131120

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

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Tegel, HannaUhlén, MathiasHober, Sophia

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