Lyophilized Escherichia Coli -Based Cell-Free Systems for Robust, High-Density, Long-Term Storage

Abstract

Cell-free protein synthesis (CFPS) is a versatile tool for rapid recombinant protein production and engineering. One drawback of cell-free technology is the necessity to store the major components-cell extracts and energy systems-below freezing in bulky aqueous solutions. Here we describe simple methods for lyophilizing extracts and preparing powdered energy systems for CFPS. These techniques allow for high-density storage of cell-free systems that are more robust against temperature and bacterial degradation. Our methods have the potential to decrease storage expenses, allow for longer shelf-life of cell extracts at room temperature, and enable durable portable protein production technologies.

The growing impact of lyophilized cell-free protein expression systems

“…Onconase yields from lyophilized extracts compared favorably with those from standard extracts, matching or exceeding the standard extract Onconase yields. Taken with our previous results, which showed extract viability after up to 90 days storage at room temperature, 25 these results demonstrate that a difficult-to-express therapeutic could be produced on-demand, even in remote locations using lyophilized extracts stored at sub-optimal conditions.…”

“…24 Inspired by this report and seeking to overcome scale-up and RNA stabilization challenges of this system, we created a lyophilized cell-free protein synthesis system based on E. coli cell extracts for shelf-stable storage and transportation. 25 The system, in addition to breaking the cold-storage chain, has the added benefits of 1) a justadd-water C DNA format where protein can be produced rapidly on-demand -in as little as 1 h, 2) consistent scalable production from 250 mL up to 100 L 26 , and 3) sterilization of the system to prevent release of residual genetically modified bacteria if transported or used in-field. 27 Thus shelf-stable, on-demand protein synthesis platforms are enabling new possibilities in biotechnology applications.…”

Establishing a high yielding streptomyces‐based cell‐free protein synthesis system

“…Cell-free protein synthesis (CFPS) is one of the most important applications of cell-free systems. The ability to freezedry CFPS systems is opening the way to novel on demand biomanufacturing applications as well (Dudley et al, 2016;Pardee et al, 2016b;Salehi et al, 2016;Smith et al, 2014). So far, they have been widely utilized for manufacturing a wide variety of active protein products that include therapeutic vaccines (Kanter et al, 2007;Yang et al, 2005), antibodies (Min et al, 2016;Stech and Kubick, 2015), viruslike particles (Bundy et al, 2008;Lu et al, 2015), membrane proteins (Henrich et al, 2015;Sachse et al, 2014), metalloproteins (Boyer et al, 2008;Kwon et al, 2013;Li et al, 2016) and proteins harboring non-standard amino acids (Hong et al, 2014(Hong et al, , 2015.…”

Creating a completely “cell‐free” system for protein synthesis

“…To create high‐yielding extracts, lysis is best accomplished by physical methods, predominantly using high‐pressure homogenization or sonication . These methods can be extremely efficient, exceeding 99.999% lysis of cells . Repeated lysis treatments increases lysis efficiency, however, increased treatment can damage the activity of the extract …”

“…For industrial applications, residual bacterial contamination can exponentially bloom and be deleterious in any scale‐up or bioreactor conditions . For commercial biodiagnostics, contamination can impact consistency and shelf‐life of the system . Furthermore, some promising applications of CFPS include portable and personalized technologies, such as pharmacy‐ and lab‐on‐a‐chip .…”