Development of freeze-dried cell protein synthesis (CFPS) kits, Syntheasy

• Grant number: 19/16625-6
• Support Opportunities: Research Grants - Innovative Research in Small Business - PIPE
• Start date: July 01, 2020
• End date: March 31, 2021
• Field of knowledge: Biological Sciences - Biochemistry
• Principal Investigator: Natalia Marchesan Bexiga
• Grantee: Natalia Marchesan Bexiga
• CNAE: Pesquisa e desenvolvimento experimental em ciências físicas e naturais ; Atividades de serviços de complementação diagnóstica e terapêutica
• Associated researchers: Marco Antonio Stephano
• Associated scholarship(s): 20/08131-0 - Development of freeze-dried cell protein synthesis (CFPS) kits, Syntheasy, BP.PIPE; 20/08590-5 - Development of freeze-dried cell protein synthesis (CFPS) kits, Syntheasy, BP.TT

Abstract

The Syntheasy " kit components, produced and marketed by BioLinker Synthetic Biology, LTDA (CNPJ 31.021.329 / 0001-62) for in vitro recombinant protein production (CFPS), are composed of highly temperature sensitive biological materials. CFPS is a technique used for in vitro protein synthesis. This technology makes it possible to flex and reduce the time spent on protein production at both the basic and applied study levels and has recently been used on an industrial production scale. Through CFPS it is possible to add non-canonical amino acids, as well as toxin biosynthesis, previously considered as limitations in the process based on expression by living organisms. Cell extracts prepared for CFPS are commonly stored at -20oC or -80oC prior to use to maintain protein synthesis viability. This requires considerable capital investment in the form of low or ultra-low temperature freezers, their maintenance, emergency backup freezers, and emergency backup power. These storage considerations are particularly complicated when storing large amounts of extract for commercial applications where there is a large scale of projected production and / or production for export and shipment of the material. An additional limitation of storing CFPS extracts below freezing temperatures is the inability to easily transport them for mobile applications. One possibility for improving the efficiency and usefulness of CFPS extract storage is to lyophilize the extracts. Freeze drying of simple protein solutions is a widely used process for removing volatile liquids, leaving only one protein powder. This technique reduces storage volume and decreases the rate of protein degradation. Our proposal is that lyophilization of E. coli cell extracts similarly reduce storage volume and potentially allow longer shelf life at temperatures well above -80oC. Freeze-dried cell free systems are commercially available, and previous studies have reported. the successful synthesis of various proteins using rehydrated lyophilized extracts. However, a simple and robust method for producing a lyophilized E. coli-based cell-free system has not yet been detailed in the literature. Moreover, the impact of lyophilization and its parameters during storage at non-standard temperatures on protein synthesis performance has not been described.In our project, mannitol, sucrose, glucose, lactose, trehalose and a combination of these with different polymers will be evaluated at concentrations initially of 5% and 10% (w / v) as potential excipients and stabilizers during freeze drying of cell extracts. While sucrose, glucose, lactose and trehalose are known as stabilizers in lyophilized protein formulations, mannitol is mainly used as a bulking agent. As the residual moisture content of lyophilized formulations can also influence stability and is an important parameter to be evaluated. To determine the robustness of the formulations, different lyophilization protocols will be performed in additional experiments. We are also interested in the impact of the annealing procedure during the lyophilization process on the physicochemical and thermal properties of cell extract formulations, especially regarding the crystallization of mannitol. Finally, the relationship between properties of lyophilized formulations and their morphology will also be evaluated. Scanning electron microscopy (SEM) will be used for morphological characterization. Additionally, and if this is the case, the degree of crystallization of mannitol will be determined by the X-ray diffraction (XRD) technique. (AU)