1. The yeast cells were cultured and collected, and the volume of the compacted cells was measured before the yeast digestion buffer was resuspended in the yeast digestion buffer. All the following steps were carried out at 4 °C.

2. Resuspend the cells with 1 volume of glass bead disruption buffer.

3. Mix 2 volumes of glass bead sterilizing buffer with the cells and add 4 volumes of ice-cold pickled glass beads.

4a. When the compressed cells are rested <10 ml, transfer the cell suspension to a suitable size screw-filled centrifuge tube (suspension accounts for ≤60%~70% of the volume of the centrifuge tube), at 4 °C to maximum Shake for 30~60 s at speed, place the tube on ice for 1~2 min, repeat 3~5 times, check the degree of cell destruction under the microscope, and continue with step 5.

4b. When the volume of compressed cells is >10 ml, transfer the cell suspension to a suitable size glass bead stirred container (suggested stainless steel with good thermal conductivity) and add glass bead sterilizing buffer to the edge of the container, but The buffer added to the container does not exceed the volume of one cell suspension. Install the stir bar and screw cap to ensure that all the air in the container is drained (it is very important to exclude air to prevent foaming and protein denaturation), stir at high speed for 60 s, place on ice for 1~2 min, repeat 3~5 times .

5. Precipitate the glass beads, gently pour out the supernatant, add 2~4 volumes of glass bead sterilizing buffer, and invert the tube 5~10 times. After the glass beads are precipitated, gently pour out the supernatant and combine the supernatant.

6. The combined supernatant was centrifuged at 12 000 g for 60 min at 4 ° C, and the supernatant was collected as an extract. For long-term storage, it was divided into small portions and rapidly frozen in liquid nitrogen, and stored at -80 °C.