Production of functional inclusion bodies in endotoxin-free Escherichia coli

Rueda, Fabián; Cano‐Garrido, Olivia; Mamat, Uwe; Wilke, Kathleen; Seras‐Franzoso, Joaquin; García‐Fruitós, Elena; Villaverde, Antonio

doi:10.1007/s00253-014-6008-9

Cited by 45 publications

(33 citation statements)

References 44 publications

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“…The controlled production of functional IB analogues in Pichia pastoris [88] and in the food-grade bacterium Lactococcus lactis has just been demonstrated [89]. Together with the recent description of IB fabrication in endotoxin-free E. coli [90,91], the production of IBs in microorganisms other than Gram-negative, endotoxin-containing bacteria is expanding the catalogues of cell factories for biologically safer IB-related products, what would conduct to a smoother implementation of IB-based protein delivery technologies in different fields.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Bacterial Inclusion Bodies: Discovering Their Better Half

Rinas

García‐Fruitós

Corchero

et al. 2017

Trends in Biochemical Sciences

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Bacterial inclusion bodies are functional, non-toxic amyloids occurring in recombinant bacteria that show analogies with secretory granules of the mammalian endocrine system. The scientific interest in these mesoscale protein aggregates has been historically masked by their status as a hurdle in recombinant protein production.However, insights into their molecular organization and the progressive understanding on how the cell handles the quality of recombinant polypeptides have stimulated the interest on inclusion bodies and opened ways for their usability in diverse technological fields. The engineering and tailoring of inclusion bodies as functional protein particles for materials science and biomedicine is a good example of how formerly undesired bacterial by-products can be re-discovered as promising functional materials for a broad spectrum of applications.-2 - BACKGROUND

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Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Bacterial Inclusion Bodies: Discovering Their Better Half

Rinas

García‐Fruitós

Corchero

et al. 2017

Trends in Biochemical Sciences

Self Cite

151

123

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“…The ClearColi extract preparation procedure differs slightly from that of standard extract due to the altered characteristics of the ClearColi cells. ClearColi cells are reportedly more sensitive to osmolarity, grow at approximately half the rate of regular BL21 cells, and reach lower final densities . Gene expression in bacteria changes dramatically with growth rate and cell phase, making induction and harvest points important parameters when preparing extract for CFPS .…”

Section: Resultsmentioning

confidence: 99%

“…Three different media formulations and varying levels of NaCl were tested: LB‐Miller (the recommended media for ClearColi protein preparations), 2xYT media (reported to produce high‐yielding CFPS extracts), and TB media (which supports E. coli growth to higher cell densities) . The three media formulations were evaluated at increasing NaCl levels, as enhanced ClearColi growth has been suggested at higher osmolarity and ionic strength .…”

Section: Resultsmentioning

confidence: 99%

Endotoxin-Free E. coli- Based Cell-Free Protein Synthesis: Pre-Expression Endotoxin Removal Approaches for on-Demand Cancer Therapeutic Production

et al. 2018

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Approximately one third of protein therapeutics are produced in Escherichia coli, targeting a wide variety of diseases. However, due to immune recognition of endotoxin (a lipid component in the E. coli cell membrane), these protein products must be extensively purified before application to avoid adverse reactions such as septic shock. E. coli-based cell-free protein synthesis (CFPS), which has emerged as a promising platform for the development and production of enhanced protein therapeutics, provides a unique opportunity to remove endotoxins prior to protein expression due to its open environment and the absence of live cells. Pre-expression endotoxin removal from CFPS reagents could simplify downstream processing, potentially enabling on-demand production of unique protein therapeutics. Herein, three strategies for removing endotoxins from E. coli cell lysate are evaluated: Triton X-114 two-phase extraction, polylysine affinity chromatography, and extract preparation from genetically engineered, endotoxin-free ClearColi cells. It is demonstrated that current protocols for endotoxin removal treatments insufficiently reduce endotoxin and significantly reduce protein synthesis yields. Further, the first adaptation of ClearColi cells to prepare cell-free extract with high protein synthesis capability is demonstrated. Finally, production of the acute lymphoblastic leukemia therapeutic crisantaspase from reduced-endotoxin extract and endotoxin-free ClearColi extract is demonstrated.

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“…Being insufficient to generate inflammation at the injection site (Figure C), this is still an issue to be considered during further IB development. However, since functional IBs or IB‐like particles have been successfully produced in endotoxin‐free strains of E. coli , in food‐grade lactic acid bacteria, and also in yeasts, the future clinically oriented development of secretory amyloids must necessarily consider these safer cell factories, favorable for the production of protein‐based drugs from strategic and regulatory point of views. Considering that any IB‐forming polypeptide with therapeutic value can also be engineered to acquire specific cell targeting, as in the case of T22‐PE24‐H6, the implantable material can be designed for precision medicines such as in cancer treatments.…”

Section: Reduction Of Metastatic Foci Induced By Pe24 Ibs Treatmentmentioning

confidence: 99%

Engineering Secretory Amyloids for Remote and Highly Selective Destruction of Metastatic Foci

et al. 2019

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Functional amyloids produced in bacteria as nanoscale inclusion bodies are intriguing but poorly explored protein materials with wide therapeutic potential. Since they release functional polypeptides under physiological conditions, these materials can be potentially tailored as mimetic of secretory granules for slow systemic delivery of smart protein drugs. To explore this possibility, bacterial inclusion bodies formed by a self‐assembled, tumor‐targeted Pseudomonas exotoxin (PE24) are administered subcutaneously in mouse models of human metastatic colorectal cancer, for sustained secretion of tumor‐targeted therapeutic nanoparticles. These proteins are functionalized with a peptidic ligand of CXCR4, a chemokine receptor overexpressed in metastatic cancer stem cells that confers high selective cytotoxicity in vitro and in vivo. In the mouse models of human colorectal cancer, time‐deferred anticancer activity is detected after the subcutaneous deposition of 500 µg of PE24‐based amyloids, which promotes a dramatic arrest of tumor growth in the absence of side toxicity. In addition, long‐term prevention of lymphatic, hematogenous, and peritoneal metastases is achieved. These results reveal the biomedical potential and versatility of bacterial inclusion bodies as novel tunable secretory materials usable in delivery, and they also instruct how therapeutic proteins, even with high functional and structural complexity, can be packaged in this convenient format.

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Production of functional inclusion bodies in endotoxin-free Escherichia coli

Abstract: Escherichia coli is the workhorse for gene cloning and production of soluble recombinant proteins in both biotechnological and biomedical industries. The bacterium is also a good producer of several classes of protein-based self-assembling materials 3

Cited by 45 publications

References 44 publications

Bacterial Inclusion Bodies: Discovering Their Better Half

Bacterial Inclusion Bodies: Discovering Their Better Half

Endotoxin-Free E. coli- Based Cell-Free Protein Synthesis: Pre-Expression Endotoxin Removal Approaches for on-Demand Cancer Therapeutic Production

Engineering Secretory Amyloids for Remote and Highly Selective Destruction of Metastatic Foci

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