Exploring the use of leucine zippers for the generation of a new class of inclusion bodies for pharma and biotechnologic
- PDF / 1,670,044 Bytes
- 13 Pages / 595.276 x 790.866 pts Page_size
- 58 Downloads / 136 Views
crobial Cell Factories Open Access
RESEARCH
Exploring the use of leucine zippers for the generation of a new class of inclusion bodies for pharma and biotechnological applications Ramon Roca‑Pinilla1, Sara Fortuna2, Antonino Natalello3, Alejandro Sánchez‑Chardi4,5, Diletta Ami3, Anna Arís1* and Elena Garcia‑Fruitós1*
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 stud‑ ied. 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 physicochemi‑ cal 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 carbo‑ hydrate 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 nanoparti‑ cles 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. Keywords: Inclusion bodies, Aggregation, Recombinant protein, Leucine zippers, Jun, Fos, Purity
*Correspondence: [email protected]; [email protected] 1 Department of Ruminant Production, Institute of Agriculture and Food Research and Technology (IRTA), 08140 Caldes de Montbui, Spain Full list of author information is ava
Data Loading...
