Upregulation of YciM Expression Reduces Endotoxin Contamination of Recombinant Proteins Produced in Escherichia coli Cells

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Pavel A. Bobrovsky^1,2, Daria D. Kharlampieva¹, Sergey A. Kirillin¹, Ksenia A. Brovina^1,2, Ekaterina N. Grafskaia¹, Vassili N. Lazarev^1,2, and Valentin A. Manuvera^1,2,a*

¹Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435 Moscow, Russia

²Moscow Institute of Physics and Technology (National Research University), 141701 Moscow, Russia

^* To whom correspondence should be addressed.

Received June 29, 2023; Revised August 10, 2023; Accepted August 10, 2023

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Recombinant proteins produced in Escherichia coli are often contaminated with endotoxins, which can be a serious problem for their further application. One of the possible solutions is the use of modified strains with reduced lipopolysaccharide (LPS) levels. We compared two approaches to engineering such strains. The first commonly known approach was modification of LPS biosynthesis pathway by knocking out seven genes in the E. coli genome. The second approach, which has not been previously used, was to increase expression of E. coli protein YciM. According to the published data, elevated expression of YciM leads to the reduction in the amount of the LpxC enzyme involved in LPS biosynthesis. We investigated the impact of YciM coexpression with eGFP on the content of endotoxins in the purified recombinant eGFP samples. Both approaches provided similar outcomes, i.e., decreased the endotoxin levels in the purified protein samples.

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KEY WORDS: recombinant protein, lipopolysaccharide, endotoxin, expression strain, lipid IVa, CRISPR, Cas9

DOI: 10.1134/S0006297923090110

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