Exploring the use of leucine zippers for the generation of a new class of inclusion bodies for pharma and biotechnological applications
Author
Roca-Pinilla, Ramon
Fortuna, Sara
Natalello, Antonino
Sánchez-Chardi, Alejandro
Ami, Diletta
Arís, Anna
Publication date
2020-09-04ISSN
1475-2859
Abstract
Background
Inclusion bodies (IBs) are biologically active protein aggregates forming natural nanoparticles with a high stability and a slow-release behavior. Because of their nature, IBs have been explored to be used as biocatalysts, in tissue engineering, and also for human and animal therapies. To improve the production and biological efficiency of this nanomaterial, a wide range of aggregation tags have been evaluated. However, so far, the presence in the IBs of bacterial impurities such as lipids and other proteins coexisting with the recombinant product has been poorly studied. These impurities could strongly limit the potential of IB applications, being necessary to control the composition of these bacterial nanoparticles. Thus, we have explored the use of leucine zippers as alternative tags to promote not only aggregation but also the generation of a new type of IB-like protein nanoparticles with improved physicochemical properties.
Results
Three different protein constructs, named GFP, J-GFP-F and J/F-GFP were engineered. J-GFP-F corresponded to a GFP flanked by two leucine zippers (Jun and Fos); J/F-GFP was formed coexpressing a GFP fused to Jun leucine zipper (J-GFP) and a GFP fused to a Fos leucine zipper (F-GFP); and, finally, GFP was used as a control without any tag. All of them were expressed in Escherichia coli and formed IBs, where the aggregation tendency was especially high for J/F-GFP. Moreover, those IBs formed by J-GFP-F and J/F-GFP constructs were smaller, rougher, and more amorphous than GFP ones, increasing surface/mass ratio and, therefore, surface for protein release. Although the lipid and carbohydrate content were not reduced with the addition of leucine zippers, interesting differences were observed in the protein specific activity and conformation with the addition of Jun and Fos. Moreover, J-GFP-F and J/F-GFP nanoparticles were purer than GFP IBs in terms of protein content.
Conclusions
This study proved that the use of leucine zippers strategy allows the formation of IBs with an increased aggregation ratio and protein purity, as we observed with the J/F-GFP approach, and the formation of IBs with a higher specific activity, in the case of J-GFP-F IBs. Thus, overall, the use of leucine zippers seems to be a good system for the production of IBs with more promising characteristics useful for pharma or biotech applications.
Document Type
Article
Document version
Published version
Language
English
Subject (CDU)
57 - Biological sciences in general
573 - General and theoretical biology
576 - Cellular and subcellular biology. Cytology
579 - Microbiology
61 - Medical sciences
615 - Pharmacology. Therapeutics. Toxicology
Pages
13
Publisher
BMC
Is part of
Microbial Cell Factories
Citation
Roca-Pinilla, Ramon, Sara Fortuna, Antonino Natalello, Alejandro Sánchez-Chardi, Diletta Ami, Anna Arís, and Elena Garcia-Fruitós. 2020. "Exploring The Use Of Leucine Zippers For The Generation Of A New Class Of Inclusion Bodies For Pharma And Biotechnological Applications". Microbial Cell Factories 19 (1). doi:10.1186/s12934-020-01425-x.
Program
Producció de Remugants
This item appears in the following Collection(s)
- ARTICLES CIENTÍFICS [2850]
Except where otherwise noted, this item's license is described as http://creativecommons.org/licenses/by/4.0/