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Automated production of recombinant human proteins as resource for proteome research.

Kohl T, Schmidt C, Wiemann S, Poustka A, Korf U - Proteome Sci (2008)

Bottom Line: The target proteins are encoded by functionally uncharacterized open reading frames (ORF) identified by the German cDNA consortium.A robust automated strategy for the production of recombinant human proteins in E. coli was established based on a set of four different protein expression vectors resulting in NusA/His, MBP/His, GST and His-tagged proteins.Future applications might include the optimization of experimental conditions for the large-scale production of soluble recombinant proteins from libraries of open reading frames.

View Article: PubMed Central - HTML - PubMed

Affiliation: German Cancer Research Center, Heidelberg, Germany. kohl@froggo.de

ABSTRACT

Background: An arbitrary set of 96 human proteins was selected and tested to set-up a fully automated protein production strategy, covering all steps from DNA preparation to protein purification and analysis. The target proteins are encoded by functionally uncharacterized open reading frames (ORF) identified by the German cDNA consortium. Fusion proteins were produced in E. coli with four different fusion tags and tested in five different purification strategies depending on the respective fusion tag. The automated strategy relies on standard liquid handling and clone picking equipment.

Results: A robust automated strategy for the production of recombinant human proteins in E. coli was established based on a set of four different protein expression vectors resulting in NusA/His, MBP/His, GST and His-tagged proteins. The yield of soluble fusion protein was correlated with the induction temperature and the respective fusion tag. NusA/His and MBP/His fusion proteins are best expressed at low temperature (25 degrees C), whereas the yield of soluble GST fusion proteins was higher when protein expression was induced at elevated temperature. In contrast, the induction of soluble His-tagged fusion proteins was independent of the temperature. Amylose was not found useful for affinity-purification of MBP/His fusion proteins in a high-throughput setting, and metal chelating chromatography is recommended instead.

Conclusion: Soluble fusion proteins can be produced in E. coli in sufficient qualities and microg/ml culture quantities for downstream applications like microarray-based assays, and studies on protein-protein interactions employing a fully automated protein expression and purification strategy. Future applications might include the optimization of experimental conditions for the large-scale production of soluble recombinant proteins from libraries of open reading frames.

No MeSH data available.


Related in: MedlinePlus

Influence of fusion tag and induction temperature on fusion protein yield. Successfully purified human fusion proteins sorted according to fusion tag and purification strategy. Protein expression was induced at 25°C (white), 30°C (dark grey) and 37°C (light grey), respectively.
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Figure 3: Influence of fusion tag and induction temperature on fusion protein yield. Successfully purified human fusion proteins sorted according to fusion tag and purification strategy. Protein expression was induced at 25°C (white), 30°C (dark grey) and 37°C (light grey), respectively.

Mentions: The influence of the different fusion tags was examined (Figure 3) and compared with the outcome of our manual approach. With respect to the impact of the induction temperature on His-tagged protein expression, 15% (14 proteins), 19% (18 proteins), 5% (5 proteins) of His-tag proteins were purified when induced at a temperature of 25°C, 30°C, and 37°C, respectively. For reasons of technical simplicity, a one-step lysis and purification procedure was performed in the automated approach. This one-step procedure monitored exclusively the successfully purified proteins without analyzing the percentage of inducible proteins. Moreover, with an average yield of close to 30%, His-tagged fusion proteins were slightly better soluble when protein expression was induced in the manual approach [30].


Automated production of recombinant human proteins as resource for proteome research.

Kohl T, Schmidt C, Wiemann S, Poustka A, Korf U - Proteome Sci (2008)

Influence of fusion tag and induction temperature on fusion protein yield. Successfully purified human fusion proteins sorted according to fusion tag and purification strategy. Protein expression was induced at 25°C (white), 30°C (dark grey) and 37°C (light grey), respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266735&req=5

Figure 3: Influence of fusion tag and induction temperature on fusion protein yield. Successfully purified human fusion proteins sorted according to fusion tag and purification strategy. Protein expression was induced at 25°C (white), 30°C (dark grey) and 37°C (light grey), respectively.
Mentions: The influence of the different fusion tags was examined (Figure 3) and compared with the outcome of our manual approach. With respect to the impact of the induction temperature on His-tagged protein expression, 15% (14 proteins), 19% (18 proteins), 5% (5 proteins) of His-tag proteins were purified when induced at a temperature of 25°C, 30°C, and 37°C, respectively. For reasons of technical simplicity, a one-step lysis and purification procedure was performed in the automated approach. This one-step procedure monitored exclusively the successfully purified proteins without analyzing the percentage of inducible proteins. Moreover, with an average yield of close to 30%, His-tagged fusion proteins were slightly better soluble when protein expression was induced in the manual approach [30].

Bottom Line: The target proteins are encoded by functionally uncharacterized open reading frames (ORF) identified by the German cDNA consortium.A robust automated strategy for the production of recombinant human proteins in E. coli was established based on a set of four different protein expression vectors resulting in NusA/His, MBP/His, GST and His-tagged proteins.Future applications might include the optimization of experimental conditions for the large-scale production of soluble recombinant proteins from libraries of open reading frames.

View Article: PubMed Central - HTML - PubMed

Affiliation: German Cancer Research Center, Heidelberg, Germany. kohl@froggo.de

ABSTRACT

Background: An arbitrary set of 96 human proteins was selected and tested to set-up a fully automated protein production strategy, covering all steps from DNA preparation to protein purification and analysis. The target proteins are encoded by functionally uncharacterized open reading frames (ORF) identified by the German cDNA consortium. Fusion proteins were produced in E. coli with four different fusion tags and tested in five different purification strategies depending on the respective fusion tag. The automated strategy relies on standard liquid handling and clone picking equipment.

Results: A robust automated strategy for the production of recombinant human proteins in E. coli was established based on a set of four different protein expression vectors resulting in NusA/His, MBP/His, GST and His-tagged proteins. The yield of soluble fusion protein was correlated with the induction temperature and the respective fusion tag. NusA/His and MBP/His fusion proteins are best expressed at low temperature (25 degrees C), whereas the yield of soluble GST fusion proteins was higher when protein expression was induced at elevated temperature. In contrast, the induction of soluble His-tagged fusion proteins was independent of the temperature. Amylose was not found useful for affinity-purification of MBP/His fusion proteins in a high-throughput setting, and metal chelating chromatography is recommended instead.

Conclusion: Soluble fusion proteins can be produced in E. coli in sufficient qualities and microg/ml culture quantities for downstream applications like microarray-based assays, and studies on protein-protein interactions employing a fully automated protein expression and purification strategy. Future applications might include the optimization of experimental conditions for the large-scale production of soluble recombinant proteins from libraries of open reading frames.

No MeSH data available.


Related in: MedlinePlus