Casein hydrolysate

Protein yields of de novo synthesized ¹⁴C-leucine labeled Nbs were determined by liquid scintillation counting as described previously (Stech et al., 2012 (link)). In brief, 5 µL aliquots of SN1 or SN2 were precipitated in 3 ml of 10% (v/v) TCA—2% (v/v) casein hydrolysate (Carl Roth GmbH, Karlsruhe, Germany), boiled for 15 min at 80°C and subsequently cooled on ice for at least 30 min. Protein solutions were filtered using a vacuum filtration system (Hoefer, Holliston, United States) and concentrations of ¹⁴C-leucine labeled proteins which were retained on the membrane filters (VWR, Darmstadt, Germany) were calculated based on liquid scintillation counting using the Hidex SL600 scintillation counter.

EtOAc (used for fungal extraction) was purchased from VWR International GmbH, Hannover, Germany. UPLC grade methanol, acetonitrile and water used for UPLC/MS analysis were purchased from BiosolveChimie, Dieuze, France. Formic acid (UPLC/MS optigrade) was obtained from LGC Standards Promochem©, Wesel, Germany. UPLC-QToF-MS/MS analyses were carried out on an ACQUITY UPLC I-Class System coupled to the Xevo G2-XS QToF Mass Spectrometer (Waters^®, Milford, MA, USA). Czapek broth, yeast extracts and malt extracts were purchased from BD Bioscience, Sparks, NE, USA. Agar was purchased from Applichem, Darmstadt, Germany. Peptone from soymeal and glucose were purchased from Merck, Darmstadt, Germany. Potato extract was from Sigma-Aldrich, Schnelldorf, Germany. Sucrose was purchased from Handelsmarken, Offenburg, Germany. Casein hydrolysate was purchased from Carl Roth, Karlsruhe, Germany.

Total protein yields of cell-free synthesized, ¹⁴C-labeled protein were determined by hot trichloroacetic acid (TCA) precipitation and subsequent liquid scintillation counting (Stech et al., 2012 (link)). In short, 3 µl of the fraction analyzed were mixed with 3 ml 10% TCA (Carl Roth GmbH & Co. KG; Karlsruhe, Germany) with 2% casein hydrolysate (Carl Roth GmbH & Co. KG; Karlsruhe, Germany) and incubated in a water bath at 80°C for 15 min followed by 30 min incubation on ice. The TCA solution was then transferred to membrane filters (VWR International GmbH, Darmstadt, Germany) using a vacuum filtration device (Hoefer, Inc., Holliston, USA). The filters were washed with 5% TCA and dried with acetone. The dry filters were transferred into scintillation vials (Sarstedt AG & Co KG) and incubated in 3 ml scintillation cocktail for 1 h. The samples were then measured using the Hidex 600 SL (Hidex; Turku, Finland). Protein yields were calculated based on the counted disintegrations per minute, the specific activity of ¹⁴C-leucine in the cell-free reaction, the molecular weight, and the number of leucines in the protein.