title: 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.
authors.affiliation: Programa de Engenharia Química - COPPE - Universidade Federal do Rio de Janeiro (UFRJ) - Av. Horácio Macedo 2030, Bloco G, Sala 115 - Centro de Tecnologia (CT) - Cidade Universitária, Ilha do Fundão, Caixa Postal 68502 - 21941-972, Rio de Janeiro, RJ, Brazil. karen@peq.coppe.ufrj.br
authors.forename: Karen
authors.initials: K
authors.lastname: Einsfeldt
authors.forename: João Baptista
authors.initials: JB
authors.lastname: Severo Júnior
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Corrêa Argondizzo
authors.forename: Marco Alberto
authors.initials: MA
authors.lastname: Medeiros
authors.forename: Tito Lívio Moitinho
authors.initials: TL
authors.lastname: Alves
authors.forename: Rodrigo Volcan
authors.initials: RV
authors.lastname: Almeida
authors.forename: Ariane Leites
authors.initials: AL
authors.lastname: Larentis
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D015870:Gene Expression; D005822:Genetic Vectors; D042822:Genomic Instability; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000378:metabolism*; D007612:Kanamycin / Q000494:pharmacology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012641:Selection, Genetic; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D007612:Kanamycin; D012697:Serine Endopeptidases; C501427:ClpP protein, Streptococcus pneumoniae; D049071:Endopeptidase Clp