Recombinant Protein Expression in <i>Rhodococcus</i> species

Producing recombinant bacterial proteins is essential for elucidating their structure and function, making efficient overexpression systems a key requirement. Escherichia coli is widely used for this purpose, particularly for bacterial proteins, but certain targets remain challenging to express or obtain as soluble protein. A recently developed Rhodococcus expression system has successfully produced proteins that were poorly expressed or not expressed in E. coli, highlighting the value of standardized protocols for this alternative host. Here, we present detailed methods for cloning into Rhodococcus expression vectors, transforming electrocompetent Rhodococcus cells, and performing protein expression and purification in this system. We also outline strategies to improve yields and provide troubleshooting guidance. These protocols are designed to equip protein scientists across disciplines with a robust alternative for bacterial protein production. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Cloning into a Rhodococcus expression vector Basic Protocol 2: Generation of electrocompetent Rhodococcus cells Basic Protocol 3: Transformation into a Rhodococcus host Basic Protocol 4: Recombinant protein production and purification using the Rhodococcus system.