Cloning and expression of protease ClpP from Streptococcus pneumoniae in Escherichia coli: Study of the influence of kanamycin and IPTG concentration on cell growth, recombinant protein production and plasmid stability

Abstract

Infections caused by Streptococcus pneumoniae are one of the main causes of death around the world. In order to address this problem, investigations are being made into the development of a protein-based vaccine. The aims of this study were to clone and express ClpP, a protein from S. pneumoniae serotype 14 in Escherichia coli, to optimize protein expression by using experimental design and to study plasmid segregation in the system. ClpP was cloned into the pET28b vector and expressed in E. coli BL21 Star (DE3). Protein expression was optimized by using central composite design, varying the inducer (IPTG) and kanamycin concentration, with a subsequent analysis being made of the concentration of heterologous protein, cell growth and the fraction of plasmid-bearing cells. In all the experiments, approximately the same concentration of ClpP was expressed in its soluble form, with a mean of 240.4mg/L at the center point. Neither the IPTG concentration nor the kanamycin concentration was found to have any statistically significant influence on protein expression. Also, higher IPTG concentrations were found to have a negative effect on cell growth and plasmid stability. Plasmid segregation was identified in the system under all the concentrations studied. Using statistical analysis, it was possible to ascertain that the procedures for determining plasmid stability (serial dilution and colony counting) were reproducible. It was concluded that the inducer concentration could be reduced tenfold and the antibiotic eliminated from the system without significantly affecting expression levels and with the positive effect of reducing costs.

Enhanced Production of Functional Extracellular Single Chain Variable Fragment Against HIV-1 Matrix Protein fromEscherichia coliby Sequential Simplex Optimization

“…The E. coli from different strains responded differently with IPTG. [32] The previous reports and our study were associated with the importance of IPTG concentration. This parameter incorporated cells density and the recombinant protein yield.…”

Experimental design approach in recombinant protein expression: determining medium composition and induction conditions for expression of pneumolysin from Streptococcus pneumoniae in Escherichia coliand preliminary purification process

“…This phenomenon could be explained by the toxic characteristic of IPTG, which is widely reported in literature [31,37]. Noteworthy, expression of other recombinant proteins from S. pneumoniae studied in our laboratory (PsaA and ClpP protease) , using this same host system, showed a negative effect of IPTG on cell growth [7,31]. …”

“…Finally, kanamycin concentration showed no statistical effect on this response, as it happened when it was evaluated in recombinant ClpP protein expression [31]. …”

Production of heterologous cutinases by E. coli and improved enzyme formulation for application on plastic degradation

“…Another aspect to be considered is the growth inhibition of these cultures because of the presence of IPTG itself. The addition of inducers is an important variable in the production of proteins, especially on a large scale, since IPTG is costly and it can be toxic to cells (Einsfeldt et al 2011). Pan et al (2008) confirmed that IPTG used to induce expression of cloned genes is a toxic compound that in high concentrations drastically reduces cell growth in some cultures of recombinant E. coli.…”