Escherichia coli-based cell extract is a vital component of inexpensive and high-yielding cell-free protein synthesis reactions. However, effective preparation of E. coli cell extract is limited to high-pressure (French press-style or impinge-style) or bead mill homogenizers, which all require a significant capital investment. Here we report the viability of E. coli cell extract prepared using equipment that is both common to biotechnology laboratories and able to process small volume samples. Specifically, we assessed the low-capital-cost lysis techniques of: (i) sonication, (ii) bead vortex mixing, (iii) freeze-thaw cycling, and (iv) lysozyme incubation to prepare E. coli cell extract for cell-free protein synthesis (CFPS). We also used simple shake flask fermentations with a commercially available E. coli strain. In addition, RNA polymerase was overexpressed in the E. coli cells prior to lysis, thus eliminating the need to add independently purified RNA polymerase to the CFPS reaction. As a result, high-yielding E. coli-based extract was prepared using equipment requiring a reduced capital investment and common to biotechnology laboratories. To our knowledge, this is the first successful prokaryote-based CFPS reaction to be carried out with extract prepared by sonication or bead vortex mixing.
Cell-free protein synthesis (CFPS) is an in vitro protein transcription/translation system in which cell maintenance and growth is detached from protein synthesis. CFPS enables direct control and optimization of protein synthesis by performing the reaction in a test tube wherein the transcription, translation, and protein folding machinery provided by cell extract are combined with energy sources to catalyze the synthesis of only the target protein. Hence, viable cell extract is a vital constituent of effective cell-free reactions, and cell lysis is a key unit operation in cell extract preparation. Due to the superior control and direct engineering that CFPS affords over protein synthesis, many independent researchers have developed, simplified and optimized CFPS reactions and cell extract preparation procedures (1-10). However, methods for inexpensive and high-yielding Escherichia coli-based CFPS still require specialized cell lysis equipment, resulting in a significant capital investment. In this work, we assess the use of cell lysis techniques with common biotechnology equipment requiring a smaller capital investment to prepare viable E. coli-based CFPS extract.
CFPS is an open system devoid of a membrane barrier and is thus amenable to direct engineering. CFPS has been of great use when toxic proteins (11,12), virus-like particles (12-14), membrane proteins (15-17), and proteins containing unnatural amino acids (18-21) are to be produced. Additionally, CFPS compliments other applications including high-throughput functional genomics (22), protein evolution (23-25), and structural proteomics and genomics (17, 26). The open nature of the CFPS allows for manifold manipulations of the system, including adjustment of energy, cofactor, and genetic template concentrations, as well as the cell extract itself. For example, many different energy sources such as phosphoenolpyruvate (27), phosphocreatine (28, 29), glucose (30, 31), and fructose-1,6 bisphosphate (32) have been successfully incorporated into CFPS, and E. coli central metabolism and oxidative phosphorylation have been activated (33, 34). Additionally, PCR-generated linear DNA templates have been incorporated in CFPS, mitigating the labor- and time-intensive processes of gene cloning (22, 35, 36). To enable more scientist and engineers to reap the benefits that CFPS has to offer, a simple, robust, convenient, and high-yielding cell extract preparation method is needed.
The E. coli-based system is the least expensive, the highest yielding, and the most time efficient CFPS system (37). The E. coli extract preparation protocol for CFPS dates back to an article from Nirenberg in 1963 (10), which was further modified by Zubay (2) and Pratt (3). More recently, Kigawa et al. (4), Liu et al. (5), Kim et al. (6), and Yang et al. (7) have sought to streamline the extract preparation protocol. Kim et al. (6) eliminated unnecessary steps and reduced the reagent cost and processing time for extract preparation by up to 80% when compared with the protocol established by Pratt (3). In addition, Kigawa et al. (4) and Yang et al. (7) have reported the use of shake flask fermentation to simplify the cell growth. Kim et al. (6) also reported the use of the commercial BL21 (DE3) strain (Invitrogen, Carlsbad, CA, USA) to overexpress the T7 RNA polymerase during cell extract preparation and eliminate the need to add independently purified T7 RNA polymerase to the CFPS reaction as required by other protocols (4, 5, 7). More recently, the same research laboratory reported the use of a BL21 Star (DE3; Invitrogen) strain that contains an RNA stabilizing mutation and will be used in this work (38). Figure 1 provides an overview of these developments. As shown in Figure 1, all of the aforementioned protocols use a specialized bead mill or high-pressure homogenizer (French press-style or impinge-style) for cell disruption, requiring a significant capital investment before research laboratories can assess the efficacy of E. coli-based CFPS for their protein of interest or application. Inspired by the successful use of freeze-thaw cycling for insect-based CFPS (8) and an unsuccessful attempt at using sonication for E. coli-based CFPS (4), here we explore more capital-cost-effective cell lysis techniques for E. coli-based CFPS. We also will explore streamlining the extract preparation method by combining the simple shake flask fermentation with the use of the commercial BL21 Star (DE3) E. coli strain and the highly simplified lysate processing protocol reported by Kim et al. (6).