Vacuum filtration system

Based on the incorporation of ¹⁴C-leucine in cell-free synthesized proteins, the respective protein yield can be estimated by scintillation measurement. Therefore, 5 µL aliquots of each translation mixture were mixed with 3 mL of a 10% (v/v) TCA-2% (v/v) casein hydrolysate solution (Carl Roth, Karlsruh, Germany) in a glass tube and incubated at 80 °C for 15 min. Afterwards, samples were chilled on ice for 30 min and retained on the surface of glass fiber filter papers (MN GF-3, Machery-Nagel, Düren, Germany) using a vacuum filtration system (Hoefer, Kleinblittersdorf, Germany). Filters were washed twice with 5% TCA and dried with acetone. Dried filters were placed into a scintillation vial (Zinsser analytic, Eschborn, Germany), 3 mL of scintillation cocktail (Quicksafe A, Zinsser analytic) was added and vials were agitated on an orbital shaker for at least 1 h. The scintillation signal was determined using the LS6500 Multi-Purpose scintillation counter (PerkinElmer, Berlin, Germany). The protein concentration was identified based on the obtained scintillation counts and protein specific parameters including molecular mass and amount of leucine. Error bars calculated for the protein yield show the individual standard deviation.

Aliquots of 5 µl were taken after 3 h reaction time, mixed with 3 ml trichloroacetic acid and incubated in 80 °C water bath for 15 min. After incubation on ice for 30 min, non-incorporated ¹⁴C-leucine was removed from the protein solution by a filtration step using a vacuum filtration system (Hoefer) as described previously¹⁸ . Measurement of incorporated ¹⁴C-leucine was performed by liquid scintillation counting (LS6500 Multi-Purpose scintillation counter, Beckman Coulter).

Yields of ¹⁴C-leucine labeled proteins were determined in TM, SN1 after centrifugation (16,000 × g, 10 min, 4 °C) and MF after resuspension of microsomes in phosphate-buffered saline (PBS, pH 7.4). To release translocated target proteins from the lumen of the microsomal vesicles, microsomes were resuspended in detergent containing buffer (0.2% DDM in PBS) and incubated under rigorous mixing for 45 min at RT. Subsequently, solutions were centrifuged and the resulting supernatant (SN2) was applied for further analysis. Following cell-free protein synthesis and fractionation into SN1, MF and SN2, aliquots of 3 µL were withdrawn from the solution, mixed with 3 mL trichloroacetic acid (TCA) and incubated in a 80 °C water bath for 15 min, followed by incubation on ice for 30 min. In order to remove non-incorporated ¹⁴C-leucine, protein solutions were filtered using a vacuum filtration system (Hoefer). Incorporation of ¹⁴C-leucine in cell-free expressed proteins was measured by liquid scintillation counting using the LS6500 Multi-Purpose scintillation counter (Beckman Coulter).

At the indicated incubation time aliquots of 5 µl were withdrawn from the cell-free translation reaction, mixed with 3 ml trichloroacetic acid and incubated in a 80°C water bath for 15 min, followed by incubation on ice for 30 min. In order to remove non-incorporated ¹⁴C-leucine from the translation mixture, protein solutions were filtered using a vacuum filtration system (Hoefer). Incorporation of ¹⁴C-leucine in cell-free expressed proteins was measured by liquid scintillation counting using the LS6500 Multi-Purpose scintillation counter (Beckman Coulter).

Determination of total protein yields was performed in triplicates based on liquid scintillation as described previously (Stech et al., 2017 (link)). Following cell-free protein synthesis and fractionation into SN1 and SN2, aliquots were withdrawn from the solution, mixed with 3 ml trichloroacetic acid (TCA), and incubated in an 80°C water bath for 15 min, followed by incubation on ice for 30 min. In order to remove non-incorporated ¹⁴C-leucine, protein solutions were filtered using a vacuum filtration system (Hoefer, Holliston, MA, United States) and glass fiber filters (Machery-Nagel, Düren, Germany). Incorporation of ¹⁴C-leucine in cell-free synthesized proteins was measured by liquid scintillation counting using the HIDEX 600 SL (Hidex, Turku, Finland).