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We're expressing a protein in a model organism for crystallization, however it seems the protein has some toxic effect on the cells.

Is there something we can do to make it non-functional during expression so it won't affect production, in such a way that we can restore function later (ex. after purification) in a simple way?

I know there are other approaches available but I'd like some inputs on this one alone.

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    $\begingroup$ That would depend on the specific protein being expressed. You could try secreting the protein or targeting it to inclusions and then refolding. $\endgroup$
    – canadianer
    Apr 17 '18 at 15:48
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    $\begingroup$ Can you provide more information? Are you expressing the protein in yeast or bacteria? $\endgroup$
    – user22542
    Apr 17 '18 at 16:00
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    $\begingroup$ I assume the toxicity is limited and not lethal. Is the protein showing the toxic effect being secreted during production or retained in the cells? $\endgroup$
    – user22542
    Apr 18 '18 at 21:05
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    $\begingroup$ With limited knowledge and information, but intrigued with the question, I can only hazard to ask if it might be possible to alter the pH of the media (or another parameter) enough to render the protein temporarily "non-functional" or at least "less toxic" while maintaining a permissive environment for the growth of the production yeast? Beer becomes a questionable environment yet the yeast don't seem to mind to a point. $\endgroup$
    – user22542
    Apr 19 '18 at 11:40
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    $\begingroup$ You need to add more details or else the question would be considered too broad and most likely put on hold. $\endgroup$
    – WYSIWYG
    Apr 19 '18 at 12:55
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Inclusion body (IB) is a technique used to express proteins that are toxic to the host cells. IBs are aggregate of highly similar or the same kind of proteins in the cytoplasm formed as stress reaction, resulting biologically inactive precipitated proteins. Such stress reactions can be induced by high temperatures, pH-shifts or occur due to high feeding rates (1,2). Active proteins can be acquired later via purification and mild solubilization of inactive IBs (1).

The second method is to use special host strains. In terms of E. coli, some strains are specifically mutated and selected to withstand the expression of toxic proteins (3). e.g. C41(DE3) and C43(DE3) are developed to enhance membrane protein production (4).

Another method is to express proteins in the periplasmic space. In E.coli, you can achieve that by fusing your recombinant protein with a proper signal peptide, which targets the recombinant proteins to the periplasm via post-translational Sec-dependent pathway (3).

In addition, I also found this product list that provides several solutions for toxic protein expression, including expression in yeasts. But in that case it is similar to your situation so I am not quite sure why it doesn't work out for you.

Resources

(1) Slouka C, Kopp J, Spadiut O, Herwig C. Perspectives of inclusion bodies for bio-based products: curse or blessing?. Appl Microbiol Biotechnol. 2019;103(3):1143-1153. doi:10.1007/s00253-018-9569-1

(2) Slouka, C., Kopp, J., Hutwimmer, S. et al. Custom made inclusion bodies: impact of classical process parameters and physiological parameters on inclusion body quality attributes. Microb Cell Fact 17, 148 (2018). https://doi.org/10.1186/s12934-018-0997-5

(3) Rosano Germán L., Ceccarelli Eduardo A. Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology 5, 172 (2014). https://www.frontiersin.org/article/10.3389/fmicb.2014.00172

(4) Kwon, SK., Kim, S., Lee, DH. et al. Comparative genomics and experimental evolution of Escherichia coli BL21(DE3) strains reveal the landscape of toxicity escape from membrane protein overproduction. Sci Rep 5, 16076 (2015). https://doi.org/10.1038/srep16076

(5) https://www.neb.com/-/media/nebus/files/brochures/proteinexp_trifold_0912.pdf?rev=3ade8cf2c18b47afb7cc57113df43147&hash=7BEDC3D4DBDAE53A09EC37ADE808C450

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