Polyethyleneimine efficiently extracts recombinant cytoplasmatic green fluorescent protein produced in Escherichia coli with high purity.

J Biotechnol

Christian Doppler Laboratory for Production of Next-Level Biopharmaceuticals in E. coli, Department of Biotechnology, University of Natural Resources and Life Sciences, 1190 Vienna, Austria. Electronic address:

Published: July 2023

We used a polycationic polymer polyethyleneimine (PEI) to develop a method to extract recombinant proteins produced in the Escherichia coli (E. coli) cytosol. Compared to high pressure homogenization, commonly used to disrupt E. coli cells, our extraction method leads to higher purity of extracts. Upon addition of PEI to the cells, flocculation occurs and the recombinant protein gradually diffuses out of the PEI/cell network. While several aspects such as the E. coli strain, the cell or PEI concentration as well as the protein titer and the pH of the buffer seem to influence the extraction rate, our results show that the PEI molecule (molecular weight and structure) must be chosen appropriately for protein extraction. The method works well with resuspended cells but can also be applied directly to fermentation broths at higher PEI concentration. This extraction approach allows for effective reduction of DNA, endotoxins, and host cell proteins levels by 2-4 orders of magnitude, and drastically facilitate the subsequent downstream processing steps such as centrifugation and filtration.

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Source
http://dx.doi.org/10.1016/j.jbiotec.2023.06.001DOI Listing

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