Preparation of a cell-free translation system from a wild-type strain of Neurospora crassa

We describe the preparation of an in vitro translation system from a wild-type strain of Neurospora crassa. The system is capable of supporting efficient and faithful translation of native and in vitro transcribed eukaryotic messages. The translation products have minimal background and can be clearly analyzed by SDS-polyacrylamide gel electrophoresis. The method of preparation of the lysate is simple, fast and reproducible. The procedure should be readily applicable to other filamentous fungi.

ular mechanism of protein synthesis and control of gene expression. Unfortunately, the commercially available eukaryotic in vitro translation systems, derived from wheat germ and rabbit reticulocytes are not suitable for translation of all eukaryotic messengers. We found it impractical to use them for some Neurospora messengers hence the development of an efficient in vitro translation system (IVTS) of fungal origin appeared warranted.
Although this strain offers the advantage of a complete absence of a rigid cell wall and facile lysis, difficulties are experienced in achieving uniform growth and reproducible cell densities on account of a heterogeneous population of cells in liquid 'cultures. An IVTS from a wild type strain should be potentially more useful as those and other artifacts originating from unrelated mutations are avoided.
During the course of a study on the isolation and cloning of the gene for Neurospora pyruvate kinase (PK) it was necessary to maximize the translation of the messenger RNA for this protein. Therefore we developed a method for preparation of a lysate from wild type N. crassa cells that was effective in supporting protein synthesis with homologous and heterologous messengers.
We obtained efficient in vitro translation of the pyruvate kinase-specific messenger RNA as well as globin mRNA using this system.
In principle this method should be readily applicable toward formulation of in vitro translation systems from other filamentous fungi in general.

Preparation of cell-free extract and in vitro protein synthesis
All of the solutions used in the preparation of the lysate were autoclaved and the glassware was washed in chromic acid and treated with diethylpyrocarbonate just before use to minimize nuclease activity. Wild type N. crassa (FGSC #262) was grown in Vogel's minimal medium + 2% sucrose at 28° C, for 16 h with shaking. The mycelium was harvested on filter paper washed thoroughly and homogenized in 2.5 ml/g cells (wet wt.) of 30 mM Hepes buffer --100 mM potassium acetate --2 mM dithiothreitol, pH 7.4, with acid-washed sand in a cold mortar.
The homogenate was centrifuged for 15 min at 23,000 g at 4° C and upper two-thirds of the supernatant was collected avoiding the top lipid layer, passed through a Sephadex-G25 coarse column, equilibrated with the same buffer.
The fractions following the column void volume, with the highest absorbance at 260 nm, were pooled and frozen at -76° C in 200 µl aliquots.
For translation of exogenously added messenger RNA, 1 ml of the lysate was treated with 12 µg of micrococcal nuclease (Boehringer-Mannheim) in the presence of 1.2 mM CaCl2 by incubation at 20° C for 5 min. The reaction was stopped by addition of EGTA to a final concentration of 2.5 mM. This nuclease-treated lysate was used for protein synthesis with 1 to 5 µg RNA and optimized Mg-and K-acetate (0.35 mM and 20 mM, respectively for the RNA enriched in PK specific message) in a final volume of 25 µ1 as described for the endogenous protein synthesis.