A Rapid and Reproducible Protocol for Protein Extraction from Actinomyces v1

Actinomyces are filamentous, Gram-positive bacteria with medical and biotechnological significance, particularly as producers of enzymes and bioactive metabolites. Protein extraction from these organisms is difficult because of their slow growth, aggregation behavior, and complex cell wall composition. Rapid and reliable extraction protocols are essential for downstream biochemical and proteomic studies. Cultures were grown in starch casein nitrate (SCN) broth for 8–10 days at 30 ± 2 °C under agitation (100 rpm). Biomass was harvested by centrifugation and repeatedly washed with phosphate-buffered saline (PBS, pH 7.0) to eliminate residual media components. Two critical steps were incorporated to improve protein recovery: (i) treatment of the washed pellet with chilled acetone, which precipitates proteins and removes interfering compounds, and (ii) solubilization of the acetone-treated pellet with 1% sodium dodecyl sulfate (SDS), which disrupts the robust actinomyketal cell wall and facilitates protein release. Extracts were stored at −20 °C for short-term use and lyophilized for long-term preservation. The combined acetone precipitation and SDS solubilization strategy yielded protein extracts of sufficient purity and concentration for electrophoresis, enzyme assays, and proteomic applications. This rapid method provides a reproducible and scalable approach for protein extraction from Actinomyces, overcoming key challenges associated with filamentous bacterial growth and cell wall resilience.