Modular Platform for Recombinant Production of Highly Toxic Bacterial Proteins.

Authors

Rina Fraenkel, Inbar Cahana, Tomer Sivan, Tal Fisher, Hen Nadav, Shani Bruchim, Jenna Weisz, Noam Deouell, Shani Cheskis, Maor Shalom, Lionel Imbert, Yaara Oppenheimer-Shaanan, Asaf Levy, Netanel Tzarum

Published in

Microbial biotechnology. Volume 19. Issue 10. Pages e70453.

Abstract

Bacterial protein toxins constitute a vast, largely untapped reservoir of potent bioactive molecules for antimicrobial discovery and biotechnology. However, their intrinsic toxicity often prevents recombinant expression in microbial hosts, limiting biochemical and functional characterization. Here, we present a modular platform for the recombinant production of highly toxic bacterial proteins based on covalent fusion of polymorphic toxins (PTs) to their cognate immunity proteins (PIMs), enabling transient toxin neutralization during expression. This strategy enabled recombinant production and purification of three previously refractory toxin families, PT7, PT8 and PT9. We further streamlined the workflow by introducing intracellular protease-mediated cleavage of the toxin-immunity fusion, reducing purification from four steps to two and increasing the recovered yield of PT9 from approximately 1-2 mg/L to ~4 mg/L. Extending the approach to PT37 further showed that computational redesign of the immunity protein and fusion architecture can enable production when the native fusion is insufficient. Purified PT7 and PT8 retained nuclease activity following denaturation and refolding, while refolded PT9 was structurally validated by X-ray crystallography, demonstrating recovery of functionally and structurally competent proteins. Together, these findings establish a modular strategy for recombinant production of highly toxic bacterial proteins and facilitate their biochemical and functional characterization.

PMID:
42888866
Bibliographic data and abstract were imported from PubMed on 17 Oct 2026.

Read full publication at:
Please sign in to see all details.

Stats

1-terrible, 9-excellent. How would you rate this publication? Sign in in to submit your rating.

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.