Limits...
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

Work flow of the automated protein production strategy. Automated steps are shown in orange, steps involving manual intervention are shown in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2266735&req=5

Figure 1: Work flow of the automated protein production strategy. Automated steps are shown in orange, steps involving manual intervention are shown in blue.

Mentions: Different automated strategies are commercially available for bacterial high-throughput protein expression screening [23], or were established by different research groups [24-29]. These approaches have several drawbacks in common. For example, only a limited number of steps of the workflow are automated, leaving the challenge to integrate them into a fully automated system. The development of an automated platform for bacterial protein expression should also include DNA handling and quality control steps, as well as the production, purification and analysis of the recombinant proteins. Hence, we undertook an independent approach based on commercial robotics to set-up an improved platform for automated protein expression screening. All individual steps, including the preparation and characterization of expression clones, transformation into bacteria, picking of expression clones, growing bacterial cultures, induction of protein expression, harvesting raw protein extracts, protein affinity purification and subsequent quality control of purified proteins (Figure 1, Table 1) were performed in a multi-titer plate format and integrated in our protein production strategy. In addition, quality control steps were also included into the automated workflow. The correct insert size of the expression clones was verified by agarose gel electrophoresis, and the E-PAGE system (Invitrogen) was used to control the size and purity of affinity-purified proteins. This resulted in the development of a robust procedure which can easily be established on comparable clone picking and liquid handling equipment.


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

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

Work flow of the automated protein production strategy. Automated steps are shown in orange, steps involving manual intervention are shown in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Work flow of the automated protein production strategy. Automated steps are shown in orange, steps involving manual intervention are shown in blue.
Mentions: Different automated strategies are commercially available for bacterial high-throughput protein expression screening [23], or were established by different research groups [24-29]. These approaches have several drawbacks in common. For example, only a limited number of steps of the workflow are automated, leaving the challenge to integrate them into a fully automated system. The development of an automated platform for bacterial protein expression should also include DNA handling and quality control steps, as well as the production, purification and analysis of the recombinant proteins. Hence, we undertook an independent approach based on commercial robotics to set-up an improved platform for automated protein expression screening. All individual steps, including the preparation and characterization of expression clones, transformation into bacteria, picking of expression clones, growing bacterial cultures, induction of protein expression, harvesting raw protein extracts, protein affinity purification and subsequent quality control of purified proteins (Figure 1, Table 1) were performed in a multi-titer plate format and integrated in our protein production strategy. In addition, quality control steps were also included into the automated workflow. The correct insert size of the expression clones was verified by agarose gel electrophoresis, and the E-PAGE system (Invitrogen) was used to control the size and purity of affinity-purified proteins. This resulted in the development of a robust procedure which can easily be established on comparable clone picking and liquid handling equipment.

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