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Parallel production and verification of protein products using a novel high-throughput screening method
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.
Show others and affiliations
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.

Place, publisher, year, edition, pages
2011. Vol. 6, no 8, 1018-1025 p.
Keyword [en]
His-tag, IMAC, Micro-scale, Protein production, Protein purification, Screening
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-39519DOI: 10.1002/biot.201000430ISI: 000294108100012OAI: oai:DiVA.org:kth-39519DiVA: diva2:442118
Funder
Knut and Alice Wallenberg Foundation
Available from: 2011-09-20 Created: 2011-09-12 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
Supervisors
Note

QC 20131120

Available from: 2013-11-20 Created: 2013-11-20 Last updated: 2013-11-20Bibliographically approved
2. High-throughput protein analysis using mass spectrometry-based methods
Open this publication in new window or tab >>High-throughput protein analysis using mass spectrometry-based methods
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the field of proteomics, proteins are analyzed and quantified in high numbers. Protein analysis is of great importance and can for example generate information regarding protein function and involvement in disease. Different strategies for protein analysis and quan- tification have emerged, suitable for different applications. The focus of this thesis lies on protein identification and quantification using different setups and method development has a central role in all included papers.

The presented research can be divided into three parts. Part one describes the develop- ment of two different screening methods for His6-tagged recombinant protein fragments. In the first investigation, proteins were purified using immobilized metal ion affinity chro- matography in a 96-well plate format and in the second investigation this was downscaled to nanoliter-scale using the miniaturized sample preparation platform, integrated selective enrichment target (ISET). The aim of these investigations was to develop methods that could work as an initial screening step in high-throughput protein production projects, such as the Human Protein Atlas (HPA) project, for more efficient protein production and purification. In the second part of the thesis, focus lies on quantitative proteomics. Protein fragments were produced with incorporated heavy isotope-labeled amino acids and used as internal standards in absolute protein quantification mass spectrometry experiments. The aim of this investigation was to compare the protein levels obtained using quanti- tative mass spectrometry to mRNA levels obtained by RNA sequencing. Expression of 32 different proteins was studied in six different cell lines and a clear correlation between protein and mRNA levels was observed when analyzing genes on an individual level. The third part of the thesis involves the antibodies generated within the HPA project. In the first investigation a method for validation of antibodies using protein immunoenrichment coupled to mass spectrometry was described. In a second study, a method was developed where antibodies were used to capture tryptic peptides from a digested cell lysate with spiked in heavy isotope-labeled protein fragments, enabling quantification of 20 proteins in a multiplex format. Taken together, the presented research has expanded the pro- teomics toolbox in terms of available methods for protein analysis and quantification in a high-throughput format.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. x, 121 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:15
Keyword
Proteomics, mass spectrometry, affinity proteomics, immunoenrichment, immunoprecipitation, IMAC, screening, protein production, protein purification, ISET, quantification, SILAC, stable isotope standard, antibody validation
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-154513 (URN)978-91-7595-292-5 (ISBN)
Public defence
2014-11-14, F3, Lindstedtsvägen 26, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20141022

Available from: 2014-10-22 Created: 2014-10-21 Last updated: 2015-02-17Bibliographically approved

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