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Monitoring protein expression levels in E. coli using a high throughput approach

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Background

We have developed a high-throughput method to rapidly identify the protein constructs which are well expressed and find out which experimental factors influence their production. From a sparse matrix designed to screen between expression strains, culture media, lysis and purification buffers for each construct, the interactions among variables leading to a higher yield of soluble recombinant protein can be easily identified.

This screening is performed by a combination of small scale fermentation in deep-well blocks, cell lysis with a 24 microtips sonicator, Ni-NTA magnetic beads purification, and an automated gel capillary electrophoresis system, which allows a high-throughput and quantitative analysis of the multiple variables in one experiment.

This technique allows one to evaluate as early as possible the expression level of the constructs, narrowing down the number of constructs subsequently going through the large scale fermentation and purification module.

Results

Using a combination of robotic systems like the QIAGEN BioRobot 3000, a microsonification device (20 kHz 24 element probe from SONICS) and an Agilent ALP5100, we rapidly monitor in parallel and in a microtiter well format the level of expression in E. coli of the different protein constructs, in 15 different conditions (see Table 1). We also aim at the definition at an early stage the buffer conditions allowing protein stabilization during cell lysis and purification (figure 1).

Table 1 :
Figure 1
figure1

Quantitative analysis of a human protein purification after cell lysis and purification with 5 different buffers: The values were obtained using the ALP5100 system. The buffers 1 to 5 are described in the table1.

Author information

Correspondence to Régis Cébe.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution 2.0 International License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Keywords

  • Sparse Matrix
  • Buffer Condition
  • Capillary Electrophoresis System
  • Element Probe
  • Expression Strain