Expression of recombinant human insulin-like growth factor type 1 [rhIGF-1] in escherichia coli

Human insulin-like growth factor type 1 [hIGF-1] is a protein consisting of 70 amino acids [MW=7.6 kDa] and mainly synthesized by liver. Mecasermin [Trade name INCRELEX] is the synthetic form of the protein which is used as an effective treatment for particular disorders such as short stature, type 1 and 2 diabetes, and wound healing. Current study was aimed to investigate the expression of human insulin-like growth factor type1 in Escherichia coli [E. coli] BL21 [DE3] expression system in order to produce an active recombinant form of the protein. For the purpose of the study, firstly codon optimization was done for hIGF-1 gene, using bioinformatics databases. Then, the gene was synthesized and inserted in pET-24a vector by a cutting strategy included NdeI and BamHI-HF enzymes. In the next step, gene was run in agarose gel and purified. The constructed expression cassette was transformed into E. coli BL21 [DE3] cells through CaCl2 heat shock method. Identification and confirmation of the transformed colonies were performed using screening PCR method. Synthesis of hIGF-1 was induced by IPTG. The expression in induced strains was analyzed by SDS-PAGE and western blotting techniques. Confirmation of cloning and IGF-1 expression cassette was carried out through genetic engineering procedures. Analysis of transformed E. coli strain with SDS-PAGE and western blotting techniques confirmed that gene was expressed in host cells. Molecular weight of the expressed protein was estimated to be 7.6 kDa. hIGF-1 expression cassette for cloning and expression in E. coli was de-signed and the protein of interest was successfully induced and identified. In addition, E. coli BL21 [DE3] can be used as a suitable host for production of recombinant hIGF-1 and this technology has a potential to be localized

Association of 757 C/T polymorphismin PRODH gene with Schizophrenia in Iranian population

Evidence is emerging for the association of polymorphisms in PRODH gene and increased risk of schizophrenia. In this project, peripheral blood sampling was obtained from 175 schizophrenia patients that their diseases were confirmed by psychiatrists. 185 healthy individuals were considered as control group. The related tests were administered on the basis of PCR-RFLP. In the continuation, statistical analysis was made on the basis of obtained genotypes from two groups of healthy and patient groups using SPSS16.0 software. The administration of this project aims at investigating the hypothesis that proline dehydrogenase gene, as one of the most important genes involved in schizophrenia incidence which is proved in various populations [outside Iran], may also be involved in incidence of schizophrenia in Iran. This study has analyzed one single nucleotide polymorphism in the PRODH gene, including 757C/T in the incidence of this disease in the given statistical population. According to our results, SNP 757 C/T may be effective in incidence of the disease since P value was < 0.01. Ultimately, our results suggest that outbreak of mutation in PRODH gene in our population can be one of causes of increasing risk of Schizophrenia in this population

Isolation, purification and characterization of proline dehydrogenase from a Pseudomonas putida POS-F84 isolate

The purpose of this study was to isolate and characterize Proline Dehydrogenase [ProDH] enzyme from Iranian soil microorganisms. Isolation and screening of L-proline degradative enzymes from soil samples was carried out. The isolate was characterized by biochemical markers and 16S rRNA gene analysis. The target ProDH was purified and the effects of pH and temperature on the activity and stability were tested. Among the 150 isolates recovered from 30 soil samples, only one was characterized as Pseudomonas putida displayed the highest enzyme activity toward L-proline [2200U/l]. The enzyme of interest was identified as a ProDH and had K[m] value of 35 mM for L-proline. The molecular mass of the purified ProDH was about 40 kDa, and determined to be a monomeric protein. The N-terminal amino acid sequences of the subunit of P. putida enzyme were determined to be MLTSSLTRIIGKSGE. ProDH exhibited high activity at temperature range of 25 to 35°C and the highest activity was achieved at 30°C. It was almost stable at temperatures between 25-30°C for 2 h. The optimum pH of ProDH activity was determined in pH=8.5 and it was stable in pH range of 8.0-9.0 up to 24 h. The enzyme was purified with a yield of 8.5% and a purification factor of 37.7. Briefly, a ProDH flavonzyme was purified and characterized from a P. putida bacterium. The specificity of P. putida enzyme toward L-proline may be advantageous for the application to the L-proline analysis

Investigation of genetic association between PRODH gene and schizophrenia in Iranian population

Schizophrenia is a complicated, debilitative mental disorder. Evidence is emerging for the association of polymorphisms in PRODH gene and increased risk of schizophrenia. In the present research, we investigated relationship between of this gene and schizophrenia disease by means of a gene polymorphism using PCR-RFLP technique.150 persons suffering from acute Schizophrenia and 160 healthy persons volunteering for this project were bled. Based on intended SNP, pair of primers was designed by Oligo7 program and polymerase chain reaction [PCR] was performed by thermo cycler. Then the resulted reactive mixture was exposed to a special enzyme, which we had intended for our study. Finally, the fragments of enzyme cut were transferred on the gel [4%] and migration pattern of resulted components were compared in healthy and patient subjects, whereby obtaining genotypes of different persons in polymorphic position. We utilized SPSS 16.0 program for statistical investigation of the work and studied SNP 1945T>C and its relation with the disease in statistical population. Our findings showed a meaningful relation between the occurrence of this nucleotide mutation and its frequency in patients [given P value=0.00]. Results of this work indicate that PRODH gene can be considered to be a significant candidate in our population as a factor influencing the occurrence of Schizophrenia

effect of nitric oxide on mouse Pre-implantation embryo development and apoptosis

Clinical studies have shown that in pathological conditions such as endometriosis and reproductive tract infection [male and female] there is an activation status of macrophages that produce large quantities of nitric oxide [NO] in addition to other effector molecules. Large amounts of NO may have embryotoxic roles and produce infertility. This study was designed to evaluate the effect of different concentrations of NO on mouse pre-implantation embryo development in vitro. Mouse embryos [2-cell stage] were cultured in media containing different concentrations of sodium nitroprusside [SNP], an NO donor, or L-arginine methyl ester [L-NAME], an NO syntase [NOS] inhibitor. At the end of culture, cell apoptosis was studied by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL] technique. The results showed that development of preimplantation embryos were inhibited by high concentration of SNP [1 and 10

Affinity purification and characterization of recombinant Bacillus sphaericus phenylalanine dehydrogenase produced by pET expression vector system.

Cloning and expression of the L-phenylalanine dehydrogenase gene, from B. sphaericus in E. coli were done. The gene was cloned in the vector pET16b and transformed into E. coli BL21 [DE3]. The functional form of the L-phenylalanine dehydrogenase enzyme was purified by affinity purification techniques, taking advantage of the ability of this enzyme to bind to the nucleotide site affinity dye, Reactive Blue 4. Approximately 3 mg of highly purified recombinant enzyme was obtained from 950 mg cell pellet [wet weight]. The Relative molecular mass of the L-phenylalanine subunits was about 41 kDa by 10% SDS-PAGE. Using this method, the enzyme was obtained with a yield of 28%, and had a specific activity of 577.3 U/mg proteins, which is purified 88 times. This method was provided a facile and effective way for preparing the enzyme with a good yield that suitable for analytical purposes