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High-throughput protein purification using an automated set-up for high-yield affinity chromatography
KTH, Skolan för bioteknologi (BIO), Proteomik.
KTH, Skolan för bioteknologi (BIO), Proteomik.ORCID-id: 0000-0002-4858-8056
KTH, Skolan för bioteknologi (BIO), Proteomik.ORCID-id: 0000-0003-0605-8417
KTH, Skolan för bioteknologi (BIO), Proteomik.
2006 (Engelska)Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 46, nr 2, s. 173-178Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2006. Vol. 46, nr 2, s. 173-178
Nyckelord [en]
high-throughput, protein purification, IMAC, recombinant protein, proteomics
Identifikatorer
URN: urn:nbn:se:kth:diva-15606DOI: 10.1016/j.pep.2005.12.010ISI: 000236828100001OAI: oai:DiVA.org:kth-15606DiVA, id: diva2:333648
Anmärkning
QC 20100525Tillgänglig från: 2010-08-05 Skapad: 2010-08-05 Senast uppdaterad: 2020-01-10Bibliografiskt granskad
Ingår i avhandling
1. Characterization of Antigenic Properties and High Throughput Protein Purification
Öppna denna publikation i ny flik eller fönster >>Characterization of Antigenic Properties and High Throughput Protein Purification
2010 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

 

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 2010. s. 90
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2010:19
Nyckelord
high throughput, protein purification, sample preparation, antigen solubility, immunogenicity, epitope mapping, antigen design
Nationell ämneskategori
Industriell bioteknik
Forskningsämne
SRA - Molekylär biovetenskap
Identifikatorer
urn:nbn:se:kth:diva-25841 (URN)978-91-7415-768-0 (ISBN)
Disputation
2010-11-12, The Svedbergsalen, Roslagstullsbacken 21, AlvaNova, Stockholm, 10:00 (Svenska)
Opponent
Handledare
Anmärkning
QC 20101102Tillgänglig från: 2010-11-02 Skapad: 2010-11-02 Senast uppdaterad: 2010-11-02Bibliografiskt granskad

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Uhlén, MathiasHober, Sophia

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Steen, JohannaUhlén, MathiasHober, SophiaOttosson, Jenny
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Proteomik
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Protein Expression and Purification

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