The Biological Potential Hidden in Inclusion Bodies

Abstract

Inclusion bodies (IBs) are protein nanoclusters obtained during recombinant protein production processes, and several studies have demonstrated their potential as biomaterials for therapeutic protein delivery. Nevertheless, IBs have been, so far, exclusively sifted by their biological activity in vitro to be considered in further protein-based treatments in vivo. Matrix metalloproteinase-9 (MMP-9) protein, which has an important role facilitating the migration of immune cells, was used as model protein. The MMP-9 IBs were compared with their soluble counterpart and with MMP-9 encapsulated in polymeric-based micelles (PM) through ionic and covalent binding. The soluble MMP-9 and the MMP-9-ionic PM showed the highest activity values in vitro. IBs showed the lowest activity values in vitro, but the specific activity evolution in 50% bovine serum at room temperature proved that they were the most stable format. The data obtained with the use of an air-pouch mouse model showed that MMP-9 IBs presented the highest in vivo activity compared to the soluble MMP-9, which was associated only to a low and a transitory peak of activity. These results demonstrated that the in vivo performance is the addition of many parameters that did not always correlate with the in vitro behavior of the protein of interest, becoming especially relevant at evaluating the potential of IBs as a protein-based nanomaterial for therapeutic purposes.

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

“…Since most of the recombinant proteins form IBs when overexpressed in bacteria [ 3 ], and some of them are only produced in this insoluble format, these nanoparticles have been broadly used as a source to obtain the soluble form of a wide range of protein of interest [ 32 ]. On the other hand, by being protein nanoparticles rich in functional recombinant protein, IBs have also been explored as a new class of biomaterial with promising applications in biocatalysis, tissue engineering, and human and animal therapies [ 7 – 9 , 11 ]. However, although different aggregation tags have been used to promote their formation, the impact of these aggregation domains in the quality of protein aggregates has not been addressed so far.…”

“…The presence of native proteins forming such aggregates has prompted to assess their potential as biomaterials for a wide range of applications, including biocatalysis, tissue engineering, and antimicrobial and cancer therapies [ 7 – 10 ]. They offer important advantages over their soluble counterparts such as high stability [ 11 ], slow-release behavior [ 12 , 13 ], and production through cost-effective processes [ 14 ]. Aiming to improve the production and biological efficiency of this nanomaterial, the increase of the aggregation tendency of proteins of interest is a key aspect and has been evaluated through the use of different aggregation tags, being VP1 [ 6 ], GFIL8 [ 15 ] and ELK16 [ 16 ] three representative examples.…”

Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

“…At present, increasing evidence show that IBs have amyloidlike structure and comprise aggregated as well as native folded proteins with preserved biological activity [2]. This fact, together with the mechanical stability and high porosity of the IBs has defined them as unconventional functional materials with a wide spectrum of applications in biotechnology and biomedicine [5][6][7]. Recently, first report towards in vitro preparation of tailored and chemically defined IBs with potential for clinical application was published [8].…”

Nucleic Acids in Inclusion Bodies obtained from E. coli Cells Expressing Human Interferon-Gamma

“…At present, increasing evidence show that IBs have amyloid-like structure and comprise aggregated as well as native folded proteins with preserved biological activity [2]. This fact, together with the mechanical stability and high porosity of the IBs has defined them as unconventional functional materials with a wide spectrum of applications in biotechnology and biomedicine [5,6,7]. Their clinical potential, however, is hindered by their undefined heterogeneous composition and the presence of hazardous contaminants from the bacterial cell, especially endotoxins [8].…”