Exploring the use of leucine zippers for the generation of a new class of inclusion bodies for pharma and biotechnological applications

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.

Tailoring the Properties of Self-Assembled Carbonic Anhydrase Supraparticles for CO2 Capture

“…In general, the active inclusion bodies can achieve significant increases in thermostability and reusability. However, the catalytic efficiency, like most immobilized enzymes, is lower than that of the free ones …”

“…However, the catalytic efficiency, like most immobilized enzymes, is lower than that of the free ones. 18 The third obstacle is the relatively poor catalytic performances (for example, low efficiency at high temperatures) of CA. Currently, engineering of CAs is a common approach to cope with this problem, among which the most representative are site-specific mutations and directed evolution.…”

The Poly-Histidine Tag H6 Mediates Structural and Functional Properties of Disintegrating, Protein-Releasing Inclusion Bodies

“…This strategy has been applied in wound healing [ 30 ], for subcutaneous [ 31 ] or intranasal delivery of antigens [ 32 , 33 ], or for the local [ 34 ] or remote [ 35 ] delivery of antitumoral proteins, namely pro-apoptotic factors or tumor-targeted microbial toxins, respectively. In veterinary medicine, they have been proved as excellent immunomodulators and nanostructured vaccines [ 32 , 36 , 37 , 38 , 39 , 40 ], since minor amounts of bacterial cell wall lipopolysaccharides from the producing bacterial cells, associated to these protein granules, probably have a positive role on the immunoprotection reached [ 41 ].…”

Cheese-whey permeate improves the fitness of Escherichia coli cells during recombinant protein production

“…After 8 h of induction with the three different inducers, we observed a downshift of the band at ~ 1639 cm −1 to ~ 1634 cm −1 , accompanied by an increase in the intensity of the band at ~ 1694 cm −1 . These spectral features, typical of GFP [ 44 ], indicate that the three different inducers cause GFP overexpression. To study its production kinetics, we therefore followed the ratio of the band at 1634 cm −1 (β-sheet) to that at ~ 1658 cm −1 (α-helix/random coil) as a function of induction times (Fig.…”