Efficient cell-free translation from diverse human cell types

Abstract

Cell-free translation systems are indispensable for studying protein synthesis, enabling researchers to explore translational regulation across different cell types. The difficulties in producing cell-free translation systems from different cell types limit the ability to study regulatory mechanisms that depend on different biological contexts. Here, we present a scalable method for preparing translation-competent lysates from a range of frequently used human cell lines using dual centrifugation. We optimized lysis conditions for adherent and suspension cells, producing high-quality lysates from HEK-293 (adherent and in suspension), HeLa, SH-SY5Y, and U2OS cells. Our results demonstrate that cell-specific factors influence translation efficiency, with adherent HeLa cells showing the highest activity. We also observed that sensitivity to lysis conditions varies between cell lines, underscoring the importance of fine-tuning parameters for efficient protein production. Our method provides a robust and adaptable approach for generating cell-type-specific lysates, broadening the application of in vitro translation systems in studying translational mechanisms.

Keywords: cell-free translation; cell-type-specific lysates; dual centrifugation; in vitro translation; protein synthesis; synthetic biology; translational regulation.

Figure 1

Preparation of translation-competent lysate from HEK FITR cells using DC. A, Comparison of luminescence output of HEK FITR lysates produced under different dual centrifugation (DC) conditions by Renilla luciferase assay. B, Examination of HEK FITR cell integrity after DC by Trypan Blue staining. C, Luciferase assay comparing in vitro translation efficiency of HEK FITR lysates prepared with or without 15% glycerol in the translation buffer. D, Comparison of translation efficiency between lysates from HEK FITR cells harvested by trypsinization or scraping. The cells were resuspended in a translation buffer containing 15% glycerol and lysed by DC (800 rpm, 1 min, −5 °C) after harvesting. E, Trypan Blue staining of HEK FITR cells harvested by trypsinization (top) or scraping (bottom). In (A, C and D) each dot depicts the value of an individual experiment for which the luminescence was measured three times. Mean and SD are shown. All translation reactions were performed using a lysate concentration of 1 x 10⁵ cell equivalents/μl containing 5 fmol/μl 3xFLAG-RLuc mRNA, at 37 °C for 1 h in a total volume of 25 μl of which everything was used for the Renilla luciferase assay.

Figure 2

Characterization of HEK FITR lysate. A–B, Western blot analysis and Renilla luciferase assay of time-course in vitro translation reactions using the 3xFLAG-RLuc mRNA reporter. C, Renilla luciferase assays of in vitro translation reactions with varying 3xFLAG-RLuc reporter mRNA concentrations. D–E, Western blot analysis of time-course in vitro translation reactions using 3xFLAG-HBB and SMG6-3xFLAG mRNAs, respectively. F, Renilla luciferase activity measurements of in vitro translation reactions of 3xFLAG-RLuc in HEK FITR lysates subjected to snap-freezing for 1x, 2x or 4x. G, Western blot analysis of in vitro translation reactions using a 3xFLAG-HBB reporter mRNA with or without a poly(A) tail. H, Renilla luciferase assay of in vitro translation reactions of the 5′ capped 3xFLAG-RLuc reporter in the presence of increasing levels of the free cap analogue m⁷G(5′)ppp(5′)G. I, Western blot analysis of in vitro translation reactions of a 3xFLAG-RLuc reporter mRNA in HEK FITR, HeLa S3 or rabbit reticulocyte lysate (RRL). Antibodies used for Western blot analysis are depicted at the bottom of the blots with vinculin serving as a loading control. All translation reactions were performed with 5 fmol RNA/μl and incubated for 1 h at the temperatures specified below. For (A–H) a lysate concentration of 1 × 10⁵ cell equivalents/μl was used and the reactions were incubated at 37 °C, except for H, for which the reactions were incubated at 33 °C. RRL translation reactions in (I) were performed according to the manufacturer’s instructions, using a lysate concentration of 70% and an incubation temperature of 30 °C. HeLa S3 and HEK FITR reactions for (I) were adjusted to a lysate concentration of 1.4 × 10⁵ cell equivalents/μl and were incubated at 37 °C. For (B, C, F and H), translation reactions with a total volume of 12.5 μl were prepared and used for the luciferase analysis. Each dot depicts the value of an individual experiment for which the luminescence was measured three times. Mean and SD are shown. For (A, D, and E), 25 μl translation reactions were performed of which 2.8 μl were loaded on the gel. For (G) 80 μl translation reactions were prepared of which 4 μl were loaded on the gel and the rest was used for RNA isolation for subsequent RT-qPCR analysis (Fig. S2, A and B). For (I) 50 μl translation reactions were performed of which 2 μl were loaded on the gel and 25 μl and 12.5 μl were used for luciferase analysis for Figure S2, E and D, respectively. Translation was inhibited in control samples by adding 0.1 mM cycloheximide (CHX).

Figure 3

Production of translation-competent lysates from various cell lines. A–D, Optimization of dual centrifugation (DC) conditions for lysate preparation from HEK FreeStyle, HeLa adherent, SH-SY5Y and U2OS cells, respectively. Translation efficiency was assessed by luciferase assay performed following in vitro translation of the 3xFLAG-RLuc reporter mRNA for 1 h at 37 °C. The DC conditions tested are indicated on the x-axis. E–H, Renilla luciferase assays of time-course in vitro translation of the 3xFLAG-RLuc mRNA in lysates of the different cell lines. The lysates employed were prepared under the conditions that yielded the highest translation efficiency (see A–D), with these optimal conditions noted at the top of each graph. I–L, Western blot analysis of translation reactions from (E–H). The antibodies used are depicted below each blot, with vinculin serving as a loading control. In (A-H), each dot depicts the value of an individual experiment for which the luminescence was measured three times. Mean and SD are shown. For (A–D) and (E–L), 25 and 40 μl translation reactions were performed, respectively. For (A–H) 25 μl of the translation reactions were used for luciferase assay. For (I–L) 2.8 μl of the translation reactions were loaded on the gel. All translation reactions were performed with a lysate concentration of 1 x 10⁵ cell equivalents/μl and 5 fmol 3xFLAG–RLuc mRNA/μl translation reaction. 0.1 mM cycloheximide (CHX) was used to inhibit translation in control samples.

Figure 4

Comparison of translation efficiency of lysates from different cell types. A, Renilla luciferase assay comparing the translation efficiency of the different lysates presented in this study. Each dot depicts the value of an individual experiment for which the luminescence was measured three times. Mean and SD are shown. B, Western blot analysis of the corresponding translation reactions from (A). The antibodies used are depicted below the blots, with vinculin serving as a loading control. For (A-B) in vitro translation reactions contained 5 fmol 3xFLAG-RLuc mRNA/μl and were performed at 37 °C for 1 h with a lysate concentration of 1 x 10⁵ cell equivalents/μl in a total volume of 40 μl. 0.1 mM cycloheximide (CHX) was used to inhibit translation in control samples. 15 μl of the reactions were used for the Renilla luciferase assay, and 2.3 μl were loaded on the gel for Western blot analysis. C, Schematic depiction of the workflow for preparing translation-competent lysate from various cell types created with Biorender.com.