University of Limerick
Miriyala_2023_Comparison.pdf (2.38 MB)

Comparison of wild-type and codon-optimized Streptococcal cysteine protease SpeB expression in E. coli

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posted on 2023-09-08, 07:28 authored by Navatha Miriyala

Streptococcus pyogenes or group A streptococcus (GAS) is a Gram-positive bacteria known to cause both mild and severe infections in humans. GAS strains secrete several proteases that play key roles in virulence role and pathogenesis. Among them, Streptococcal pyrogenic exotoxin B (SpeB) is the most widely studied protease that plays a key role in the virulence by degrading host proteins. SpeB is a cysteine protease reported to cleave numerous host and streptococcal proteins. The structural and functional characteristics of purified SpeB have been reported. SpeB production was initially carried out from the native GAS stains and later expanded to recombinant production in Escherichia coli cells. When expressed in E. coli using a suitable expression vector, various levels of solubilities were achieved under optimized conditions. The current study used a codon optimization strategy in combination with various induction parameters to improve the recombinant production of SpeB in E. coli cells. Both wildtype (SpeB23_wild) and codon optimized SpeB (SpeB23_co) were cloned into pET23b plasmid, expressed into E. coli BL21(DE3)pLysS cells and induced with IPTG at 20, 25 and 30oC, respectively to achieve optimal solubility. In comparison with SpeB23_wild, higher purification yield was observed with SpeB23_co when a single step Ni-NTA affinity chromatography was performed. Furthermore, SpeB23_co shown similar activity than that of SpeB23_wild in both skim-milk agar and chromogenic substrate hydrolysis-based assays. The similar activity results suggest that the codon optimization did not impair SpeB catalytic activity. In summary, the current study highlights the utilization of codon optimization in SpeB production to achieve improved yields of active protein.



  • Faculty of Science and Engineering


  • Master (Research)

First supervisor


Second supervisor

T.F. Kagawa

Other Funding information

I extend my gratitude to SFI and SSPC for the financial assistance granted to me.

Department or School

  • Biological Sciences

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