Stefano Giordania, Anna Placcia, Andrea Carbonia, Matteo Di Giosiaa, Tommaso Rossib, Barbara Rodaa,c, Andrea Zattonia,c, Pierluigi Reschigliana,c, Alberto Daniellib,c, Matteo Calvaresia, Valentina Marassia,c

aDepartment of Chemistry “Giacomo Ciamician”, University of Bologna, 40129 Bologna, Italy;
bDepartment of Pharmacy and Biotechnology, University of Bologna, 40126 Bologna, Italy;
cbyFlow srl, 40129 Bologna; Italy.

Protein nanoparticles, generated by self-assembling fusion proteins expressed by engineered bacteria, have garnered significant interest due to their potential applications in vaccine technology and biotechnology [1]. However, their production and application require efficient analytical methods for purifying and characterizing these systems. Currently, these proteins nanoparticles undergo long, multistep purification processes from the bacterial environment before characterization. This characterization typically involves costly, complex, and time-consuming techniques (e.g., TEM, HPLC-MS, SDS-PAGE), which often fail to maintain the native conditions, potentially affecting the results [1, 2]. Herein, we present a method to efficiently separate, characterize and purify bacterial lysates engineered to express a self-assembling fusion protein, namely mi3-nanobody (NB), using an AF4-DAD-MALS-FLD platform. The AF4 technique provided a gentle separation and the use of a physiological mobile phase (PBS, pH=7.4) ensured that the analyte remained unaltered during the analysis. The platform delivered orthogonal information concerning the molecular weight, size, and spectroscopic properties, thus allowing the identification of the fusion proteins, evaluation of their aggregation state, estimation of their concentration, and monitoring of the lysate evolution over time. By analysing lysates obtained with different procedures (e.g. lysis/post-lysis treatments) we assessed the impact of several parameters on the lysate content, optimizing the biotechnological protocol. Additionally, the method allowed collecting purified fractions of the separated samples for further analysis or application. The fast analysis (<40 minutes) and minimal sample need (<10 μL of lysate) make this method a promising approach for advancing the use of such systems in modern science.

Keywords: AF4, Multidetection, Self-assembly proteins, Lysate purification.

References

1. Bruun T. U., Andersson A. M. C., et al., ACS nano, 12.9 (2018), pag. 8855-8866 DOI: 10.1021/acsnano.8b02805

2. Zakeri B., Fierer J. O., et al. Proc Natl Acad Sci U S A, 109(12) (2012), pag. 690-697 DOI: 10.1073/pnas.1115485109109