Volume 5 Supplement 1

The 4th Recombinant Protein Production Meeting: a comparative view on host physiology

Open Access

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

  • Régis Cébe1 and
  • Martin Geiser1
Microbial Cell Factories20065(Suppl 1):P78

DOI: 10.1186/1475-2859-5-S1-P78

Published: 10 October 2006

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

:

 

Expression strain

Culture medium

Lysis and purification buffer

Condition 1

Strain 2

Medium 2

Buffer 1

Condition 2

Strain 3

Medium 1

Buffer 4

Condition 3

Strain 3

Medium 2

Buffer 2

Condition 4

Strain 1

Medium 1

Buffer 1

Condition 5

Strain 2

Medium 3

Buffer 3

Condition 6

Strain 1

Medium 3

Buffer 4

Condition 7

Strain 2

Medium 2

Buffer 4

Condition 8

Strain 1

Medium 2

Buffer 3

Condition 9

Strain 3

Medium 2

Buffer 5

Condition 10

Strain 1

Medium 1

Buffer 5

Condition 11

Strain 2

Medium 3

Buffer 5

Condition 12

Strain 3

Medium 1

Buffer 3

Condition 13

Strain 3

Medium 3

Buffer 1

Condition 14

Strain 2

Medium 1

Buffer 2

Condition 15

Strain 1

Medium 3

Buffer 2

Sparse matrix design (http://www.igs.cnrs-mrs.fr/samba/samba.html): Strain 1: BL21(DE3)/Strain 2: C43(DE3)(from OverExpress™)/Strain3: BL21(DE3) harboring pG-KJE8 vector from Takara Medium 1: LB/Medium 2: Auto-induction/Medium 3: Turbo broth+ Augmedium from AthenaES™ Buffer 1: 40 mM HEPES;150 mM NaCl;10%glycerol;pH7/Buffer 2: 40 mM HEPES;400 mM NaCl;100 mM urea;pH7/Buffer 3: PBS pH8/Buffer 4: 40 mM Tris;150 mM NaCl;10%glycerol;pH8.5/Buffer 5: 40 mM Tris;400 mM NaCl;100 mM urea;pH8.5

Figure 1

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.

Authors’ Affiliations

(1)
Novartis Institutes for Biomedical Research

Copyright

© Cébe and Geiser; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd.

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