ABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0156692.].
ABSTRACT
L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.
Subject(s)
Antineoplastic Agents/therapeutic use , Asparaginase/therapeutic use , Escherichia coli/metabolism , Leukemia/drug therapy , Recombinant Proteins/therapeutic use , Zymomonas/enzymology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Asparaginase/genetics , Asparaginase/pharmacology , Base Sequence , Bioreactors , Cell Cycle/drug effects , Cell Line, Tumor , Cell Nucleus Shape/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Child , Child, Preschool , Cloning, Molecular , Computer Simulation , Female , Humans , Infant , Leukemia/pathology , Male , Phylogeny , Recombinant Proteins/genetics , Recombinant Proteins/pharmacologyABSTRACT
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.
Subject(s)
Bacterial Proteins/biosynthesis , Escherichia coli/growth & development , Gene Expression , Genomic Instability , Isopropyl Thiogalactoside/metabolism , Kanamycin/pharmacology , Serine Endopeptidases/biosynthesis , Transcriptional Activation/drug effects , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Cloning, Molecular , Endopeptidase Clp , Escherichia coli/genetics , Genetic Vectors , Humans , Plasmids , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Selection, Genetic , Serine Endopeptidases/genetics , Streptococcus pneumoniae/enzymology , Streptococcus pneumoniae/geneticsABSTRACT
The polymorphic region sequences in the iap gene were analyzed in 25 strains of Listeria monocytogenes isolated from cheeses in the state of Rio Grande do Sul, and compared with reference strains. This investigation distinguished two clusters of L. monocytogenes: I (20 strains) and II (5 strains).
A seqüência da região polimórfica do gene iap foi analisada em 25 cepas de Listeria monocytogenes isoladas de queijo no Estado do Rio Grande do Sul e comparadas com cepas referências. Esta investigação distinguiu L. monocytogenes em dois grupos: I (20 cepas) e II (5 cepas).
Subject(s)
Humans , Animals , Amino Acids/analysis , Cultured Milk Products , Food Analysis , Genes , In Vitro Techniques , Listeria monocytogenes/genetics , Listeria monocytogenes/isolation & purification , Cheese/analysis , Food Samples , Genotype , MethodsABSTRACT
The polymorphic region sequences in the iap gene were analyzed in 25 strains of Listeria monocytogenes isolated from cheeses in the state of Rio Grande do Sul, and compared with reference strains. This investigation distinguished two clusters of L. monocytogenes: I (20 strains) and II (5 strains).
