Optimization of engineered-modular endolysins expression conditions in Escherichia coli NiCo21(DE3) for the control of Streptococcal infection

Abstract

Abstract Streptococci are Gram-positive bacteria which cause diverse animal and human diseases and systemic infections. Antibiotic therapy for this pathogen is often unsuccessful and contributes to the development of antibiotic resistance. Bacteriophage modular endolysins, as well as their chimeric constructs, represent promising antimicrobials with high specificity against these bacteria and unlikely to evoke antimicrobial resistance. Our previous work has developed the recombination and fusion engineered-modular endolysins design (namely EλCφ and EλCφEφ, respectively) from a particular modular endolysin EφCφ to obtain novel antimicrobial with enhanced activity. In this study, we investigated further the optimization of the engineered-modular endolysin expression conditions in Escherichia coli NiCo21(DE3). Accordingly, clone selection, final inducer concentration, and incubation temperature were optimized. To obtain a recombinant clone which exhibits optimum protein expression level, expressions of the recombinant protein were conducted in shake flask. Subsequently, the soluble and insoluble protein crude extracts were collected by lysis and denaturation treatments, respectively, and verified by SDS-PAGE and Western blot analysis. The result showed that the selected clone for optimum expression of EφCφ, EλCφ, and EλCφEφ was clone 1, 3, and 5, respectively. Moreover, the optimum final inducer concentration for both EλCφ and EφCφ was 2000 μM, whereas for EλCφEφ was 500 μM. Besides, the optimum incubation temperature for protein expression for EλCφEφ was incubation at 37ºC and followed by induction at 25ºC, and for both EλCφ and EφCφ were both incubation and induction at 25ºC. Furthermore, these three proteins were found to be expressed as insoluble proteins. Collectively, these results could contribute to the development of ‘tailor-made’ antimicrobials by modular endolysin engineering, which can be used for the control of Streptococcal infection.