Production and Molecular Design of Recombinant Proteins for Nanoparticle Functionalization and for the Protein Corona Modulation

Despite the outstanding properties of nano-size materials, nanomedical products are struggling to reach the clinic due to several complications regarding the nanoparticle’s design, the formation of the protein corona, undesired interactions with the immune system, and the orientation-controlled conjugation of biomolecules to nanomaterials. In this study, we address some of these challenges from a molecular biology and biochemical perspective in order to provide information and innovative ideas that may help the development of successful nano-formulations. First, we develop a scFv antibody containing the variable regions of the heavy and light chain of a monoclonal antibody against human Adrenomedullin (hAM). The scFv antibody against hAM could therefore hold a dual function as both therapeutic agent, blocking hAM’s pro-cancerous functions, and as targeting agent, directing conjugated nanomaterials or drugs to the tumour environment. Different scFv constructs were expressed in E. coli BL21 DE3 in the form of inclusion bodies and were recovered through an in vitro refolding. The resulting soluble scFv constructs preserve their antigen binding affinity and specificity as observed by direct ELISA. Simultaneously, we have developed a novel bioconjugation approach for the assembly of proteins onto the nanoparticle’s surface in an orientation-controlled manner. Although a plethora of conjugation techniques have already been proposed, only a few of those techniques take into account protein orientation after attachment, and they all come with certain disadvantages. The orientation of proteins on the nanoparticle’s surface is of vital importance in order to carry out their function. Here, we propose a conjugation approach based on the SpyTag-SpyCatcher technology and the maleimide chemistry. Eventually , we investigated the interaction between the protein Factor VII-Activating Protease (FSAP) and cationic gold nanoparticles. After entering a biological fluid, nanoparticles are known to acquire a layer of biomolecules – mainly proteins – called the protein corona, which has shown to determine the biological fate of the nanomaterials. We found that FSAP can bind to both cationic and anionic nanoparticles. However, the interaction with cationic nanoparticles seems to trigger FSAP’s activation in a concentration dependent manner. At lower nanoparticle concentrations, the activation of FSAP was not confined to the protein corona, but led to a generalised activation of human plasma FSAP. We created two recombinant truncated version of FSAP. Contrary to what we expected, none of the fragments were able to compete with the human plasma FSAP for its binding to cationic gold nanoparticles. In fact, the recombinant proteins were found to potentiate the activator effects of the nanoparticles. These results highlight the importance of the protein corona on the physiological effects of nanomaterials and offer some insights into the potential mechanisms of cationic gold nanoparticle’s toxicity.