TY - JOUR
T1 - Highly efficient recombinant production and purification of streptococcal cysteine protease streptopain with increased enzymatic activity
AU - Lane, Michael D.
AU - Seelig, Burckhard
PY - 2016/5/1
Y1 - 2016/5/1
N2 - 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.
AB - 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.
KW - Autoinduction
KW - Enzyme purification
KW - Maturation
KW - Protease
KW - Streptococcal pyrogenic exotoxin B
KW - Zymogen
UR - http://www.scopus.com/inward/record.url?scp=84955511579&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84955511579&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2016.01.002
DO - 10.1016/j.pep.2016.01.002
M3 - Article
C2 - 26773742
AN - SCOPUS:84955511579
VL - 121
SP - 66
EP - 72
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
ER -