Understanding the mechanism of polyethyleneimine-mediated cell disintegration and protein extraction in E. coli: The role of floc network formation and PEI molecular weight.

Cell disintegration and protein extraction are crucial steps in downstream process development for biopharmaceuticals produced in E. coli. In this study, we explored the extraction mechanism of polyethyleneimine (PEI) at the cellular level and characterized the floc network that is formed upon PEI addition by Focused Beam Reflectance Measurement and Dispersion Analyzer. PEI disintegrates the cells by detachment of the outer membrane allowing protein to diffuse into the interspace of the flocs. Protein release into the supernatant occurs by diffusion out of the floc network. We could show that the type and concentrations of PEIs with varying molecular weight determines the floc properties and thus the extraction efficiency. We could demonstrate why optimal conditions, using 70 kDa PEI at 0.25 g/g cell dry mass, lead to efficient extraction while at suboptimal conditions extraction is almost negligible. Our findings provide valuable insights into the relationship between floc properties and PEI-driven protein extraction, with potential applications in bioprocessing and biotechnology.

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

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.