Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.
Bacterial Proteins ; Diphtheria Toxin/genetics* ; Escherichia coli/genetics* ; Humans ; Molecular Chaperones/genetics* ; Recombinant Proteins/genetics*

Apoptosis ; Human Umbilical Vein Endothelial Cells ; Humans ; Lipoproteins, LDL/adverse effects* ; MicroRNAs/genetics* ; Molecular Chaperones/genetics*

Humans ; Neoplasms/genetics* ; Nuclear Proteins ; Co-Repressor Proteins ; Cell Line, Tumor ; Molecular Chaperones

OBJECTIVETo explore the feasibility of using PCR-based capillary electrophoresis method to analysis mutation of the TOR1A gene in a family affected with primary torsion dystonia (PTD).

METHODSPeripheral blood sample was collected from proband and amnionic fluid from her fetus for the extraction of DNA. The 5th exon of the TOR1A gene and its flanking sequences were amplified with PCR and analyzed with agarose electrophoresis, fluorescence labeled fragment analysis and Sanger sequencing.

RESULTSFluorescence labeled fragment analysis was performed through capillary electrophoresis, which showed that the proband carried a c.907_909delGAG (p.Glu303del) deletional mutation of the TOR1A gene. The result was verified by Sanger sequencing. The fetus DNA was also found with the same mutation by capillary electrophoresis, inferring that the fetus was probably affected with the disease.

CONCLUSIONThe mutation of c.907_909delGAG of the TOR1A gene was speculated as pathologic cause of proband in this family. Fragment analysis by capillary electrophoresis combined with DNA sequencing is an efficient test for small deletional mutations and feasible for its prenatal diagnosis.

Adult ; Dystonia ; diagnosis ; genetics ; Electrophoresis, Capillary ; Female ; Humans ; Molecular Chaperones ; genetics ; Mutation ; Prenatal Diagnosis ; Sequence Analysis, DNA

OBJECTIVETo investigate the presence of Cosmc gene mutation in children with Henoch-Schönlein purpura (HSP) and the association between Cosmc gene mutation and the susceptibility to HSP.

MESULTSEighty-four children who were diagnosed with HSP between March 2014 and December 2015 were selected as the HSP group. Fifty-eight healthy volunteers matched for age and sex were enrolled as the control group. Fasting venous blood (5 mL) from the two groups was collected in EDTA anticoagulated tubes, followed by the isolation of peripheral blood mononuclear cells (PBMCs) through density gradient centrifugation. Genomic DNA was extracted from PBMCs according to the manufacturer's protocol, and the whole exon region of Cosmc gene was amplified by touch-down polymerase chain reaction (touch-down PCR). The PCR products were identified by 1% agarose gel and sequenced in order to further examine the association between Cosmc gene mutation and the susceptibility to HSP.

RESULTSSequencing results showed two mutations (c.393T>A and c.72A>G) of Cosmc gene in children with HSP. There were no significant differences in the genotype and allele frequencies at the two loci between the HSP and control groups, and this distribution was not associated with sex.

CONCLUSIONSThe mutations c.393T>A and c.72A>G in the exon region of Cosmc gene in children with HSP are not associated with the onset of HSP.

Child ; Child, Preschool ; Female ; Genetic Predisposition to Disease ; Humans ; Male ; Molecular Chaperones ; genetics ; Mutation ; Purpura, Schoenlein-Henoch ; etiology ; genetics

Child ; Dystonia Musculorum Deformans ; diagnostic imaging ; drug therapy ; genetics ; Female ; Humans ; Male ; Molecular Chaperones ; genetics ; Mutation ; Pedigree ; Positron-Emission Tomography

OBJECTIVETo investigate the effect of hepatitis C virus (HCV) non-structure protein NS5A on the activity of calcium-regulating protein alpha subunit of nascent polypeptide-associated complex (NACA) promoter.

METHODSHepG2 cell plasmid pCAT3-NACA, containing NACA promoter, was transfected alone or cotransfected with pcDNA3.1(-)-NS5A, and chloramphenicol acetyl transferase (CAT) enzyme activity was assayed by enzyme-linked immunoassay (ELISA).

RESULTSThe CAT activity in the pcDNA3.1(-)-NS5A cotransfection group was 20.7% of the CAT activity in the pCAT3-NACA group.

CONCLUSIONHCV non-structural protein NS5A has a down-regulating effect on the promoter of NACA gene.

Base Sequence ; Down-Regulation ; Humans ; Molecular Chaperones ; biosynthesis ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics ; Transcriptional Activation ; Transfection ; Viral Nonstructural Proteins ; genetics ; pharmacology

As thermostable enzymes and organisms are much more needed, researches on heat shock proteins(HSPs) of hyperthermophilic archaea have drawn more concerns. HSPs from hyperthermophilic archaea are concise only with HSP60, sHSP, prefoldin and AAA+proteins, but without HSP100s, HSP90s, HSP70 (DnaK), HSP40 (DnaJ) and GrpE which are common in mesophilic or thermophilic archaea. Accordingly, studies on the structure, function and operation mechanism of these four groups are much more important and meaningful. This review focuses on the recent progress in the researchs on the structure, function, operation mechanism and cooperation of the HSPs from hyperthermophilic archaea. The problems and obfuscations in these HSPs are analyzed, and farther research direction and key points are put out.
Archaea ; classification ; metabolism ; Archaeal Proteins ; metabolism ; Chaperonin 60 ; metabolism ; Heat-Shock Proteins ; genetics ; metabolism ; Molecular Chaperones ; metabolism

OBJECTIVE: To explore the association between rare HSPB1 variants and amyotrophic lateral sclerosis (ALS). METHODS: We performed next-generation sequencing for 166 Chinese ALS patients to screen for possible pathogenic rare variants of HSPB1. The control individuals were obtained from 1000 Genome Project and an in-house whole-exome sequencing database. The Sequence Kernel Association Test (SKAT) and the SKAT-optimal test (SKAT-O) were used to identify the association between rare HSPB1 variants and ALS. RESULTS: We identified 3 possible pathogenic rare variants of HSPB1 (all were missenses), including c.379C>T (p.R127W), c.446A>C (p.D149A) and c.451A>C (p.T151P). Compared with 1000 Genome Project, SKAT p=3.61×10 CONCLUSIONS Rare variants of HSPB1 are probably associated with the pathogenesis of ALS.
Amyotrophic Lateral Sclerosis/genetics* ; Asian Continental Ancestry Group ; Heat-Shock Proteins ; Heterozygote ; High-Throughput Nucleotide Sequencing ; Humans ; Molecular Chaperones ; Phenotype

OBJECTIVETo investigate the frequency of GAG deletion in the DYT1 gene among early onset primary dystonia patients in China.

METHODSThirteen patients with early onset primary torsion dystonia were screened for mutation in exon 5 of the DYT1 gene using denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing, and the results were confirmed with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

RESULTSThe GAG deletion mutation which results in Glu302del in exon 5 of the DYT1 gene was found in 5 patients. The detecting results were consistent between with DHPLC and PCR-RFLP. We did not find any other mutations in the DYTI gene.

CONCLUSIONSThe GAG deletion in the DYT1 gene is common amongst early onset primary torsion dystonia patients in China. The frequency of DYT1 mutation is not significantly different between European and Asian patients with early onset primary dystonia.

Adolescent ; Adult ; Base Sequence ; Child ; China ; Chromatography, High Pressure Liquid ; DNA Primers ; Dystonia ; genetics ; Female ; Humans ; Infant ; Male ; Molecular Chaperones ; genetics ; Mutation