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Characterization of Antigenic Properties and High Throughput Protein Purification
KTH, School of Biotechnology (BIO), Proteomics.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

To understand the cellular processes, knowledge of the localization and function of proteins are essential. There are several high throughput ventures examining the human proteome. However, there are some bottlenecks in these ventures. For example the production and expression of soluble proteins for analysis. Another obstacle for affinity proteomics is the generation of high quality antibodies, invaluable tools in biotechnological applications. The objective in this thesis was to facilitate protein purification and sample preparation before analysis and downstream applications. We also aimed to attain more information on what constitutes an ideal immunogen, and on how different immune systems respond to a common amino acid sequence.

 

In one of the projects an automated purification set-up was developed to ensure high recovery of up to milligram amounts of protein with high purity. The system allowed up to 60 recombinant proteins to be purified under both native and denaturing conditions. In another project, the same developed set-up was additionally shown to work with an alternative chromatography resin with small adjustments. Instead of immobilized metal ion affinity chromatography, used in the first project, ion exchange chromatography was applied under denaturing conditions, with good results. To further automate the production line in high throughput projects, an automated sample preparation was set up for mass spectrometry and e.g. gel electrophoresis analysis. We showed that a crude cell lysate could be used as input in the magnetic bead based system, and totally absent from manual handling, the output was purified and buffer exchanged samples ready for mass spectrometry analysis, as well as a fraction of sample that could be used for complementary analyses, for example gel electrophoresis to determine the protein concentration and purity.

 

The other objective was – as noted – to gain better comprehension of antibody generation to foreign proteins, and to shed more light over how to design a good antigen. First was a solubility assay developed that determined the remaining fraction of soluble protein after reduction of the concentration of denaturing agent. The assay was performed in a 96 deep well plate, and only instrumentation available in a standard laboratory was necessary. The fact that the assay could be automated on a pipetting robot, increased the throughput and reduced the necessary manual handling. Obtained information on antigen solubility was correlated to the cognate antibody titers. At average the antibody yield was higher when a soluble antigen was used for immunization. Also, the probability of failing in eliciting an immune response was increased if an insoluble antigen was used. However, the antibody titers in each solubility class were highly diverse, and thus also some insoluble antigens were found that provoked the immune system. To further examine the differences between different B cell repertoires, a massive epitope mapping was performed with more than 400 different antisera reacting to the same amino acid sequence. Antigenic hot spot regions were discovered, as well as regions depleted in antibody recognition. However, in one third of the antisera the most abundant antigenic region did not elicit any binding of antibodies. This further validates the conclusion that good antigen design is essential, however is it not certain the outcome of immunizations can ever be determined a priori due to the variability between hosts. An alternative to immunization is selection of affinity reagents by phage display. In the last project an initial parallelized set-up selected antibody fragments that showed high specificity and were compatible with several biotechnological applications, making the set-up a promising alternative to conventional immunization in proteome-wide endeavors.

 

Place, publisher, year, edition, pages
Stockholm: KTH , 2010. , 90 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:19
Keyword [en]
high throughput, protein purification, sample preparation, antigen solubility, immunogenicity, epitope mapping, antigen design
National Category
Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
Identifiers
URN: urn:nbn:se:kth:diva-25841ISBN: 978-91-7415-768-0 (print)OAI: oai:DiVA.org:kth-25841DiVA: diva2:360129
Public defence
2010-11-12, The Svedbergsalen, Roslagstullsbacken 21, AlvaNova, Stockholm, 10:00 (Swedish)
Opponent
Supervisors
Note
QC 20101102Available from: 2010-11-02 Created: 2010-11-02 Last updated: 2010-11-02Bibliographically approved
List of papers
1. High-throughput solubility assay for purified recombinant protein immunogens
Open this publication in new window or tab >>High-throughput solubility assay for purified recombinant protein immunogens
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2005 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1752, no 1, 6-10 p.Article in journal (Refereed) Published
Abstract [en]

A high-throughput assay is described for analysis of the solubility of purified recombinant proteins. The assay is based on affinity purification of proteins in the presence of chaotropic agents followed by a dilution and incubation step to investigate the solubility in the absence of high concentrations of such agents. The assay can be performed in a 96-well format, which makes it well suited for high-throughput applications. For 125 recombinant proteins expressed as part of an antibody-based proteomics effort, experimental solubility data were compared to calculated hydrophobicity values based on the amino acid sequence of each protein. This comparison showed only weak correlation between the theoretical and experimental values, which emphasizes the importance of experimental assays to determine the solubility of recombinant proteins.

Keyword
protein solubility, high-throughput, in vitro assay, urea, proteomics, antibodies, expression, products, genomics, region
Identifiers
urn:nbn:se:kth:diva-15029 (URN)10.1016/j.bbapap.2005.07.002 (DOI)000231753500002 ()2-s2.0-23944491599 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
2. High-throughput protein purification using an automated set-up for high-yield affinity chromatography
Open this publication in new window or tab >>High-throughput protein purification using an automated set-up for high-yield affinity chromatography
2006 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 46, no 2, 173-178 p.Article in journal (Refereed) Published
Abstract [en]

One of the key steps in high-throughput protein production is protein purification. A newly developed high-yield protein purification and isolation method for laboratory scale use is presented. This procedure allows fully automated purification of up to 60 cell lysates with milligram yields of pure recombinant protein in 18.5 h. The method is based on affinity chromatography and has been set up on an instrument that utilizes positive pressure for liquid transfer through columns. A protocol is presented that includes all steps of equilibration of the chromatography resin, load of sample, wash, and elution without any manual handling steps. In contrast to most existing high-throughput protein purification procedures, positive pressure is used for liquid transfer rather than vacuum. Positive pressure and individual pumps for each liquid channel contribute to controlled flow rates and eliminate the risk of introducing air in the chromatography resin and therefore ensure stable chromatography conditions. The procedure is highly reproducible and allows for high protein yield and purity.

Keyword
high-throughput, protein purification, IMAC, recombinant protein, proteomics
Identifiers
urn:nbn:se:kth:diva-15606 (URN)10.1016/j.pep.2005.12.010 (DOI)000236828100001 ()
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
3. High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography
Open this publication in new window or tab >>High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography
2007 (English)In: Biotechnology Journal, ISSN 1860-6768, Vol. 2, 709-716 p.Article in journal (Refereed) Published
Abstract [en]

A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.

Identifiers
urn:nbn:se:kth:diva-12816 (URN)10.1002/biot.200700060 (DOI)17492715 (PubMedID)
Note
QC20100622Available from: 2010-05-12 Created: 2010-05-12 Last updated: 2010-11-02Bibliographically approved
4. Automated sample preparation method for mass spectrometry analysis on recombinant proteins
Open this publication in new window or tab >>Automated sample preparation method for mass spectrometry analysis on recombinant proteins
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2009 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1216, no 20, 4457-4464 p.Article in journal (Refereed) Published
Abstract [en]

A completely automated procedure for the purification and desalting of proteins with a polyhistidine purification tag prior to mass spectrometry analysis is presented. The system is ideal for rapid quality control and optimization studies and it provides researchers with a straightforward, reliable tool for studies of recombinant proteins. Forty-eight samples can be prepared within 4.5 h and only small cultivation and buffer volumes are needed. In this proof of concept, 19,000-35,000 Da recombinant proteins from both crude and clarified cell lysates were successfully prepared for subsequent analysis by electrospray ionization anti matrix-assisted laser desorption/ionization mass spectrometry as well as by gel electrophoresis.

Keyword
Mass spectrometry, High-throughput, Automated sample preparation, Recombinant proteins, IMAC, RPLC, affinity-chromatography, purification
Identifiers
urn:nbn:se:kth:diva-18416 (URN)10.1016/j.chroma.2009.03.041 (DOI)000265956100021 ()
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
5. The correlation between antigen solubility and immunogenicity
Open this publication in new window or tab >>The correlation between antigen solubility and immunogenicity
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In antigen design, several characteristics contribute to the final success of the antigen to elicit the desired immunogenicity. However, it is difficult to screen theses attributes due to the variability within the host immune receptor repertoire. Herein, with the massive numeral of immunizations performed within a proteome-wide endeavor to produce affinity reagents to human proteins, the correlation between the solubility of the antigens and the immunogenicity was investigated. We showed that increased solubility of the antigen resulted in higher success rate in provoking the immune defense as well as higher antibody titers. We have also shown, that the increased antibody titers after affinity purifications indeed reflectthe concentration of target specific antibodies within the serum. Finally, the amino acid composition of soluble antigens was determined to be over-represented in polar residues.

National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-25854 (URN)
Note
QC 20101102Available from: 2010-11-02 Created: 2010-11-02 Last updated: 2010-11-02Bibliographically approved
6. Antigenic mapping and characterization of Albumin Binding Protein
Open this publication in new window or tab >>Antigenic mapping and characterization of Albumin Binding Protein
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The possibility to predict the location of antigenic determinants is a desirable feature in antibody production ventures and for vaccine development. However, antigenic propensity scales available today are poor, and so far it is not possible to predict the best antigen to trigger the immune system. Here, a unique set of 411 antisera towards a common part of allantigens within the Human Protein Atlas project has made it possible to perform massive epitope mapping. This effort generated a true map of the antigenic regions of this common N-terminal tag, and rendered it possible to further investigate what features that generate a good antigen. Investigations on variation in epitope occurrence are often an obstacle when mapping antigens, because of the ethics of using more animals than necessary for antibody production. As a consequence, not much has been done to verify epitopes found and the variance between different immunizations has not been thoroughly investigated. Herein it was shown that the most immunopotentating sites were only detected by the polyclonal antibodies in 70% of the immunizations, demonstrating the need of good antigen design. Detected epitopes also showed that aromatic amino acids, some positively charged aminoacids, and serine and glycine were over-represented in the antigenic hot spot regions. The detected antigenic regions were also shown to have fairly low correlation to several antigenic propensity scales.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-25855 (URN)
Note
QC 20101102Available from: 2010-11-02 Created: 2010-11-02 Last updated: 2010-11-02Bibliographically approved
7. Surrogate antigens as targets for proteome-wide binder selection
Open this publication in new window or tab >>Surrogate antigens as targets for proteome-wide binder selection
Show others...
2011 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 28, no 4, 302-311 p.Article in journal (Refereed) Published
Abstract [en]

In the last decade, many initiatives have been taken to develop antibodies for proteome-wide studies, as well as characterization and validation of clinically relevant disease biomarkers. Phage display offers many advantages compared to conventional antibody generation by immunization and hybridoma technology, since it is an unlimited resource of affinity reagents without batch-to-batch variation and is amendable for high throughput. One of the major bottlenecks to proteome-wide binder selection is the limited supply of suitable target antigens representative of the human proteome. Here, we provide proof of principle of using easily accessible, cancer-associated protein epitope signature tags (PrESTs), routinely generated within the Human Protein Atlas project, as surrogate antigens in phage selectionsfor the retrieval of target specific binders. These binders were subsequently tested in western blot, immunohistochemistry and protein microarray application to demonstrate their functionality.

Keyword
PrEST, monoclonal antibodies, phage display
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-25857 (URN)10.1016/j.nbt.2010.12.005 (DOI)000292717500002 ()21232647 (PubMedID)2-s2.0-79958056952 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
Updated from submitted to published.Available from: 2010-11-02 Created: 2010-11-02 Last updated: 2017-12-12Bibliographically approved

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Citation style
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  • en-US
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  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
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Output format
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