A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

3. Super folder GFP with 6 histidine residues (sfGFP-HIS6) sequence: The sequence was cloned into a pET9a vector using restriction sites NdeI and BamHI.

Appendix C. Product integrity analysis:
The cell-free reaction products were analyzed for their integrity and the formation of Quantification of the resulting bands confirms that above 95% of the produced protein is the desired protein -sfGFP (27 kDa) or Tyrosinase (36 kDa). Fig A(c) presents similar trend for the control commercial CFPS system.

Appendix D. Analysis of protein aggregation formation:
The cell-free reaction products that were used for the protein integrity evaluation (Appendix C), were further analyzed for their soluble fractions using a centrifugation-based protocol [2,3]. Each sample was centrifuged at 20,000 x g for 30 min at 4 °C using MegaFuge centrifuge (Thermo Scientific). The supernatant was collected and labeled as the soluble fraction. The samples were analyzed as described previously in Appendix C.
By comparing the total to the soluble fraction, Fig A demonstrates that the aggregation propensity is protein dependent. For example, sfGFP is produced in a highly soluble form (about 95% is soluble), while tyrosinase has a large insoluble fraction (47%).

Fig A. Protein integrity and aggregation assay.
Cell-free production of sfGFP and tyrosinase using the S30-T7 CFPS system (a) & (b) or using a commercial system -the S30 T7 High-Yield Protein Expression System (Promega) (c). The reaction mixtures included biotinylated lysine-tRNA complex, which enables the detection of truncated products. The total and soluble fraction were used to estimate the aggregation formation during the cellfree reactions.

Appendix E. Fluorescence analysis of sfGFP using SDS-PAGE:
In order to demonstrate that the measured fluorescence is obtained by the full protein, SDS-PAGE analysis using fluorescent imaging was used. S30-T7 CFPS systems sourced from two strains of E. coli at different reaction temperatures (37 or 45 ˚C) were used to produce sfGFP. The produced protein was mixed with SDS-PAGE sample buffer and analyzed using electrophoresis on a 12% SDS-PAGE gel. Each gel was loaded with a protein ladder, reactions with or without sfGFP encoding plasmid and purified protein in different concentrations. The gels were visualized using ImageQuant Las4000 (GE, Sweden) according to their fluorescence after excitation with blue laser.  (Fig A(a)), where a single band was also obtained without the detection of truncated products. A secondary band is observed due to secondary folding of the protein under mild denaturation conditions [4].  -4), cell-free reaction without a DNA template (lanes 5-7) and purified protein (lanes 8-10 with 3.1 µg, 1.6 µg and 0.8 µg protein, respectively). The primary band indicates that the fluorescence of the functional protein corresponds to the full length product at all reaction conditions. A secondary band is observed due to secondary folding of the protein under mild denaturation conditions [4].

Appendix F. Pseudomonas exotoxin A production and purification:
E. coli BL21 transformed with the PE plasmid (nucleotide sequence in Appendix A.5) glycerol stock (-80 C) was streaked on Luria Bertani (LB) plate solidified with 1.5 % Bacto agar, supplemented with ampicillin at 100 µg ml -1 (LB-amp100) to maintain the plasmids. A single colony was used to inoculate fresh LB-amp100 media. The culture was grown overnight at 37 °C while shaking at 250 rpm on a TU-400 incubator shaker (Orbital Shaker Incubator, MRC, Holon, Israel) and used as a starter to inoculate fresh Super Broth media (supplemented with 100 µg ml -1 ampicillin) the following day at 1:100 starter:medium ratio.
The culture was grown at 37 °C to OD600≈ 2.5, upon which 1 mM IPTG was added. The culture was further grown overnight at 30 °C and then centrifuged at 5,000 x g for 15 min at 4 °C using MegaFuge centrifuge (Thermo Scientific). The pellet was gently resuspended using sterile glass beads in ice cold 20% sucrose, 30 mM Tris-HCl (pH 7.4), 1 mM EDTA (1:5 buffer to original growth media volume), and left on ice for 15 min. Cells were then centrifuged for 15 min at 6000 rpm (FIBRLITE F15-6x100y rotor, Thermo Scientific) at 4 °C.
The pellet was gently resuspended in ice cold sterile double-distilled water (1:5 buffer to original growth media volume), and left on ice for 15 min. Following incubation on ice, the periplasmic fraction was collected by centrifuging the cells for 15 min at 7000 rpm and 4 °C (FIBRLITE F15-6x100y rotor, Thermo Scientific). The resulting periplasmic fraction was adjusted to 20 mM Tris-HCl (pH 7.4). The sample was then applied to a Q-sepharose anion exchange column (HiTrap-1ml, GE Healthcare) and purified using fast protein liquid chromatography (FPLC, AKTA, GE). The buffers used for purification were 20 mM Tris HCl pH 7.4 (Buffer A), used for equilibration and washing, and 1 M NaCl in Buffer A (Buffer B), used as the elution buffer. A gradient of Buffer B from 0-100% over 10 min was used in order to purify the PE. Eluted protein was collected and dialyzed against PBS. E. coli BL21 that was not transformed with a plasmid was produced and purified as described above and the parallel elution fractions were used as a negative control. All the elution fractions were analyzed using an SDS-PAGE 12% gel and Coomassie blue staining. Proteins eluted after 6 min by the anion exchange column were used as the control for further investigations. Original Western blot analysis of Pseudomonas exotoxin productions. S30-T7 CFPS system originated from two different E. coli strains (BL21 and MRE600) and a commercial system (S30 T7 High-Yield Protein Expression System, Promega) were used for the different protein productions. Reactions were performed with and without the presence of DNA template. The yellow frame indicates on the production of Pseudomonas exotoxin A. ~ 66 kDa, when a DNA template was incorporated to the reaction. The lower bands are not representing a 66 kDa protein and are related to the S30 extract. They can be contributed to unspecific reactivity of the polyclonal antibodies used in this analysis.