Characterisation of a Gram-positive secreted protein signal sequence (SPss) : localisation and processing during transport in a Gram-negative bacterium

Abstract

Secreted proteins offer the potential to reduce purification cost to enzyme and therapeutic proteins. While many bacterial transport signal sequences have been tested, few have been used across species. This study explores the effectiveness of a Gram-positive secreted protein signal sequence (SPss) used in a Gram-negative production platform (Escherichia. coli BL21(DE3)). A pET bacterial expression plasmid was constructed with a secreted protein signal sequence (SPss) from a secreted protein originating in Clostridium perfringens and combined with a common marker protein (Green Fluorescent Protein). The construct (SPss-GFP) was expressed in E. coli and tested under a variety of temperatures (25°C, 30°C, 37°C), IPTG concentrations (1μM, 10μM, 100μM, and 1000μM), and two growth media (Lysogeny Broth and an enzyme-release complex medium) to ascertain the ideal expression conditions. The results indicated that the SPss-GFP protein was present in the periplasm after expression, but transport was sporadic with most experiments showing only cytoplasmic GFP. More periplasmic protein was present when a control plasmid, expressing the full-length secreted iv protein from C. perfringens was used. The parameters that resulted in the highest levels of GFP was 100μM IPTG at 30°C grown in the enzyme-release medium. High levels of expression and transport were also observed at 1000μM, 37°C and at 1000μM, 30°C. Reduced cell concentration and rate of growth was observed during expression of SPss-GFP, but no parameter was found to be the cause and requires further investigation. Mass spectrophotometry indicated cleavage of SPss but much of the GFP remained in the cytoplasm. Based on the results obtained in this study, it is concluded that protein folding kinetics and limited interaction with accessory transport factors are the main problems with protein transport in this system.