A high-throughput microtiter plate-based screening method for the detection of full-length recombinant proteins.
The Gram-negative bacterium Escherichia coli is an important host for the (heterologous) production of recombinant proteins. The development and optimization of a protocol to overproduce a desired protein in E. coli is often tedious. A novel high-throughput screening method based on the Luminex xMAP bead technology was developed allowing a rapid evaluation of a certain expression strategy. A variant of green fluorescent protein (GFPuv) from Aequorea victoria was used as a reporter to establish the methodology. The N-terminus and the C-terminus of GFPuv were engineered to contain a His(6)- and an HA-tag (YPYDVPDYA), respectively. The double-tagged protein was loaded onto Luminex-microspheres via its His(6)-tag, the presence of the HA-tag was verified using an anti-HA antibody. High-throughput detection of full-length proteins (containing both tags) on the beads was performed using an automated Luminex 100IS analyzer. The results were compared to results obtained by classical Western blot analysis. Comparison of the two methods revealed that the Luminex-based method is faster and more economical in detecting full-length (intact) soluble recombinant protein, allowing one to routinely screen a high number of parameters in gene expression experiments. As proof of concept, different protocols to overproduce double-tagged model eucaryotic proteins (human protein S6 kinase 1 and human tankyrase) in E. coli were monitored using the new approach. Relevant parameters for optimizing gene expression of the corresponding genes were rapidly identified using the novel high-throughput method.
Institute for Technical Microbiology, Mannheim University of Applied Sciences, Paul-Wittsack-Str. 10, 68163 Mannheim, Germany. m.mack@hs-mannheim.de
Matthias
M
Mack
Maren
M
Burger
Patricia
P
Pietschmann
Björn
B
Hock
D015153:Blotting, Western; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008863:Microspheres; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics; D013050:Spectrometry, Fluorescence
D011994:Recombinant Proteins