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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

Quality control of recombinant fusion proteins. (A) Image of a Coomassie-stained E-PAGE gel, here shown for the purification of GST fusion proteins. (B) 96 samples can be loaded on a single E-PAGE gel comprising twelve lanes in eight rows (A-H). A single additional lane is available per row to accommodate a molecular weight standard. (C) Single lanes (each 2 cm in length) are assembled to an artificial gel image to facilitate sample analysis. (D) Example molecular weight marker separated by the E-PAGE system.
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Figure 2: Quality control of recombinant fusion proteins. (A) Image of a Coomassie-stained E-PAGE gel, here shown for the purification of GST fusion proteins. (B) 96 samples can be loaded on a single E-PAGE gel comprising twelve lanes in eight rows (A-H). A single additional lane is available per row to accommodate a molecular weight standard. (C) Single lanes (each 2 cm in length) are assembled to an artificial gel image to facilitate sample analysis. (D) Example molecular weight marker separated by the E-PAGE system.

Mentions: The liquid handling steps required for ORF cloning, protein expression and protein purification were implemented on the MULTI-probe II robot which was controlled with the application system software, if possible. Additional external equipment integrated into the robotic platform was navigated with the LabVIEW software. Clone picking was realized on the QPix robot. Figure 1 summarizes the single steps implemented into the automated routine. Open reading frames were transferred by Gateway LR reaction into four different destination vectors (Step1) and subsequently transformed into the bacterial strain DH5α for the amplification of recombinant expression plasmids (Step2). The automated restriction digest of expression plasmids confirmed the correct insert size for 361 of the 384 expression clones (Steps 3–5). Thus, 94% of destination clones were available for transformation into the bacterial strain BL21-SI (Step 6). In summary, each candidate was subjected to 15 different expression tests varying in the choice of fusion tag, induction temperature and purification strategy, or a combination thereof. Again, clone picking and the growth of pre-cultures were performed using our automated set-up (Steps 7, 8). However, the induction of protein expression by addition of IPTG or AHT is faster when performed manually (Step 9). Cultures were placed on a shaker at the indicated temperature (Step 10). Protein expression was stopped by removing the culture medium using gravity-driven filter plates. After lysis and affinity-purification (Step 11) the yield of recombination fusion proteins was analyzed using the E-PAGE system, a gel-based approach suitable for the high throughput analysis of proteins (Step 12). A single E-PAGE gel can accommodate all samples from a 96-well plate and additional molecular weight standards (Figure 2A, B). The final analysis is assisted by the E-PAGE software allowing to reassemble twelve sample lanes, corresponding to a single 96-well row, into a single image (Figure 2C). Calculation of the molecular weight of the purified fusion proteins is based on a molecular weight marker (Figure 2B, D). The yield is summarized in the Additional file 1. In order to count as successfully purified, the resulting fusion protein had to yield a clean band of the expected molecular weight. This analysis was performed using the E-PAGE system which separates proteins over a distance of merely 2 cm. The low resolution capacity of the E-PAGE system was accounted for by introducing the rule that only those proteins were regarded as successfully purified when at least two independent expression tests resulted in a protein band of the expected size. According to these criteria, 52% of the uncharacterized proteins were purified in fusion with at least one of the different tags, and quantities up to 10 μg/ml culture were obtained (Additional file 1). This yield was also reported for other strategies relying on the affinity purification of fusion proteins from small volume cultures [25,35]. However, the yield differs from our manual approach, where close to 80% of fusion proteins were obtained in quantities up to 100 μg/ml. Since the proteins analyzed in these two studies were comparable with respect to molecular weight and intracellular localization, we conclude that parameters such as aeration of culture, and the simplified one-step cell lysis and affinity purification strategy contribute to the reduced overall yield of the automated protein production strategy.


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

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

Quality control of recombinant fusion proteins. (A) Image of a Coomassie-stained E-PAGE gel, here shown for the purification of GST fusion proteins. (B) 96 samples can be loaded on a single E-PAGE gel comprising twelve lanes in eight rows (A-H). A single additional lane is available per row to accommodate a molecular weight standard. (C) Single lanes (each 2 cm in length) are assembled to an artificial gel image to facilitate sample analysis. (D) Example molecular weight marker separated by the E-PAGE system.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Quality control of recombinant fusion proteins. (A) Image of a Coomassie-stained E-PAGE gel, here shown for the purification of GST fusion proteins. (B) 96 samples can be loaded on a single E-PAGE gel comprising twelve lanes in eight rows (A-H). A single additional lane is available per row to accommodate a molecular weight standard. (C) Single lanes (each 2 cm in length) are assembled to an artificial gel image to facilitate sample analysis. (D) Example molecular weight marker separated by the E-PAGE system.
Mentions: The liquid handling steps required for ORF cloning, protein expression and protein purification were implemented on the MULTI-probe II robot which was controlled with the application system software, if possible. Additional external equipment integrated into the robotic platform was navigated with the LabVIEW software. Clone picking was realized on the QPix robot. Figure 1 summarizes the single steps implemented into the automated routine. Open reading frames were transferred by Gateway LR reaction into four different destination vectors (Step1) and subsequently transformed into the bacterial strain DH5α for the amplification of recombinant expression plasmids (Step2). The automated restriction digest of expression plasmids confirmed the correct insert size for 361 of the 384 expression clones (Steps 3–5). Thus, 94% of destination clones were available for transformation into the bacterial strain BL21-SI (Step 6). In summary, each candidate was subjected to 15 different expression tests varying in the choice of fusion tag, induction temperature and purification strategy, or a combination thereof. Again, clone picking and the growth of pre-cultures were performed using our automated set-up (Steps 7, 8). However, the induction of protein expression by addition of IPTG or AHT is faster when performed manually (Step 9). Cultures were placed on a shaker at the indicated temperature (Step 10). Protein expression was stopped by removing the culture medium using gravity-driven filter plates. After lysis and affinity-purification (Step 11) the yield of recombination fusion proteins was analyzed using the E-PAGE system, a gel-based approach suitable for the high throughput analysis of proteins (Step 12). A single E-PAGE gel can accommodate all samples from a 96-well plate and additional molecular weight standards (Figure 2A, B). The final analysis is assisted by the E-PAGE software allowing to reassemble twelve sample lanes, corresponding to a single 96-well row, into a single image (Figure 2C). Calculation of the molecular weight of the purified fusion proteins is based on a molecular weight marker (Figure 2B, D). The yield is summarized in the Additional file 1. In order to count as successfully purified, the resulting fusion protein had to yield a clean band of the expected molecular weight. This analysis was performed using the E-PAGE system which separates proteins over a distance of merely 2 cm. The low resolution capacity of the E-PAGE system was accounted for by introducing the rule that only those proteins were regarded as successfully purified when at least two independent expression tests resulted in a protein band of the expected size. According to these criteria, 52% of the uncharacterized proteins were purified in fusion with at least one of the different tags, and quantities up to 10 μg/ml culture were obtained (Additional file 1). This yield was also reported for other strategies relying on the affinity purification of fusion proteins from small volume cultures [25,35]. However, the yield differs from our manual approach, where close to 80% of fusion proteins were obtained in quantities up to 100 μg/ml. Since the proteins analyzed in these two studies were comparable with respect to molecular weight and intracellular localization, we conclude that parameters such as aeration of culture, and the simplified one-step cell lysis and affinity purification strategy contribute to the reduced overall yield of the automated protein production strategy.

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