Exploring the use of leucine zippers for the generation of a new class of inclusion bodies for pharma and biotechnological applications
Roca-Pinilla, Ramon (Institut de Recerca i Tecnologia Agroalimentàries. Centre de Recerca en Sanitat Animal)
Fortuna, Sara (University of Trieste. Department of Chemical and Pharmaceutical Sciences)
Natalello, Antonino (University of Milano-Bicocca. Department of Biotechnology and Biosciences)
Sánchez Chardi, Alejandro (Universitat Autònoma de Barcelona. Servei de Microscòpia)
Ami, Diletta (University of Milano-Bicocca. Department of Biotechnology and Biosciences)
Arís i Giralt, Anna (Institut de Recerca i Tecnologia Agroalimentàries. Centre de Recerca en Sanitat Animal)
Garcia-Fruitos, Elena (Institut de Recerca i Tecnologia Agroalimentàries. Centre de Recerca en Sanitat Animal)

Data:	2020
Resum:	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. 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. 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.
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Inclusion bodies ; Aggregation ; Recombinant protein ; Leucine zippers ; Jun ; Fos ; Purity
Publicat a:	Microbial cell factories, Vol. 19 (september 2020) , ISSN 1475-2859

DOI: 10.1186/s12934-020-01425-x
PMID: 32887587

13 p, 1.6 MB

El registre apareix a les col·leccions:
Documents de recerca > Documents dels grups de recerca de la UAB > Centres i grups de recerca (producció científica) > Ciències de la salut i biociències > Centre de Recerca en Sanitat Animal (CReSA-IRTA)
Articles > Articles de recerca
Articles > Articles publicats