Aggregation-prone peptides modulate interferon gamma functionality in naturally occurring protein nanoparticles
Authors: Carratalá José Vicente , Cano-Garrido Olivia , Sánchez Julieta , Membrado Cristina , Pérez Eudald , Conchillo-Solé Oscar , Daura Xavier , Sánchez-Chardi Alejandro , Villaverde Antonio , Arís Anna , Garcia-Fruitós Elena , Ferrer-Miralles Neus 
Published: January 2019

AbstractEfficient protocols for the production of recombinant proteins are indispensable for the development of the biopharmaceutical sector. Approximately 400 recombinant protein-based biopharmaceuticals have been approved in recent decades, with steady growth projected in the coming years. During the expression of a heterologous gene, the protein quality control network is overcome by the disruption in protein homeostasis, leading to protein aggregation. This phenomenon has been described in all expression systems analyzed to date, including prokaryotic and eukaryotic host cells. These protein aggregates have long been considered inert protein clumps devoid of biological activity and their study has largely been neglected. However, in recent years, the classic view of protein aggregates has completely changed with the recognition that these aggregates are a valuable source of functional recombinant proteins. In this study, bovine interferon-gamma (rBoIFN-γ) was engineered to enhance the formation of protein aggregates by the addition of aggregation-prone peptides (APPs) in the generally recognized as safe (GRAS) bacterial Lactococcus lactis expression system. The L6K2, HALRU and CYOB peptides were selected to assess their intrinsic aggregation capability to nucleate protein aggregation. These APPs enhanced the tendency of the resulting protein to aggregate at the expense of the total protein yield. However, fine physicochemical characterization of the resulting intracellular protein nanoparticles (NPs), the protein released from these protein NPs, and the protein purified from the soluble cell fraction indicated that the compactability of protein conformations is directly related to the biological activity of variants of IFN-γ, which is used here as a model protein with therapeutic potential.ImportanceThe demand for recombinant proteins in the pharmaceutical industry is steadily increasing. Emerging novel protein formulations, including naturally occurring protein NPs, might be an alternative to soluble variants for fine analysis at the biophysical level. Such analyses are important to address safety about biological molecules.This study analyzes the effect of aggregation-prone peptides (APPs) on the improvement of the production of naturally occurring protein nanoparticles (NPs) of interferon gamma (IFN-γ) in the generally recognized as safe (GRAS) Lactococcus lactis expression system. In addition, the fine physico-chemical characterization of the resulting proteins, either obtained from the soluble or insoluble cell fractions, indicates that the selected engineered proteins embedded in the protein NPs show higher compactability than their soluble protein counterparts. Conformational compactability is directly related to the biological performance of the recombinant IFN-γ.