Streptococcus pyogenes produces the cysteine protease streptopain (SpeB) as a critical virulence factor for pathogenesis. Despite having first been described seventy years ago, this protease still holds mysteries which are being investigated today. Streptopain can cleave a wide range of human proteins, including immunoglobulins, the complement activation system, chemokines, and structural proteins. Due to the broad activity of streptopain, it has been challenging to elucidate the functional results of its action and precise mechanisms for its contribution to S. pyogenes pathogenesis. To better study streptopain, several expression and purification schemes have been developed. These methods originally involved isolation from S. pyogenes culture but were more recently expanded to include recombinant Escherichia coli expression systems. While substantially easier to implement, the latter recombinant approach can prove challenging to reproduce, often resulting in mostly insoluble protein and poor purification yields. After extensive optimization of a wide range of expression and purification conditions, we applied the autoinduction method of protein expression and developed a two-step column purification scheme that reliably produces large amounts of purified soluble and highly active streptopain. This method reproducibly yielded 3 mg of streptopain from 50 mL of expression culture at >95% purity, with an activity of 5306 ± 315 U/mg, and no remaining affinity tags or artifacts from recombinant expression. This improved method therefore enables the facile production of the important virulence factor streptopain at higher yields, with no purification scars that might bias functional studies, and with an 8.1-fold increased enzymatic activity compared to previously described procedures.
Bibliographical noteFunding Information:
We would like to thank Dr. Patrick Schlievert in the Department of Microbiology at the University of Iowa for the donation of the plasmid containing streptopain, Dr. Florian Seebeck for suggesting the autoinduction protocol, Dr. LeeAnn Higgins in the Center for Mass Spectrometry and Proteomics at the University of Minnesota for her assistance in the mass spectrometry studies and data analysis, Dr. Ebbing de Jong in the College of Biological Sciences at the University of Minnesota for his assistance with STAGE TIP design and production, and Dr. Matilda Newton and Dr. Yari Cabezas for their comments on the manuscript. This work was funded by the US National Institutes of Health (NIH) ( AI113406 ; MSTP grant T32 GM008244 to MDL), the Minnesota Medical Foundation , the American Heart Association (MDL), and by a grant from the Biocatalysis Initiative of the BioTechnology Institute at the University of Minnesota .
- Enzyme purification
- Streptococcal pyrogenic exotoxin B