Rapid one-step separation and purification of recombinant phenylalanine dehydrogenase in aqueous two-phase systems

Background: Phenylalanine dehydrogenase (PheDH; EC 1.4.1.20) is a NAD +-dependent enzyme that performs the reversible oxidative deamination of L-phenylalanine to phenylpyruvate. It plays an important role in detection and screening of phenylketonuria (PKU) diseases and production of chiral intermediates as well. The main goal of this study was to find a simple and rapid alternative method for purifying PheDH. Methods: The purification of recombinant Bacillus sphaericus PheDH was investigated in polyethylene glycol (PEG) and ammonium sulfate aqueous two-phase systems (ATPS). The influences of system parameters including PEG molecular weight and concentration, pH and (NH4)2SO4 concentration on enzyme partitioning were also studied. The purity of enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Results: A single extraction process was developed for separation and purification of recombinant PheDH from E. coli BL21 (DE3). The optimized conditions for partitioning and purification of PheDH were 9% (w/w) PEG-6,000 and 16% (w/w) (NH4)2SO4 at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were achieved 58.7, 135%, 94.42%, 491.93 and 9828.88 U/mg, respectively. Also, the Km values for L-phenylalanine and NAD+ in oxidative deamination were 0.21 and 0.13 mM, respectively. Conclusion: The data presented in this paper demonstrated the potential of ATPS as a versatile and scaleable process for downstream processing of recombinant PheDH

Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR

Darbepoetin alfa is an engineered and hyperglycosylated analog of recombinant human erythropoietin (EPO) which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. This study desribes the secretory expression of a codon-optimized recombinant form of darbepoetin alfa in Leishmania tarentolae T7-TR. Synthetic codon-optimized gene was amplified by PCR and cloned into the pLEXSY-I-blecherry3 vector. The resultant expression vector, pLEXSYDarbo, was purified, digested, and electroporated into the L. tarentolae. Expression of recombinant darbepoetin alfa was evaluated by ELISA, reverse-transcription PCR (RT-PCR), Western blotting, and biological activity. After codon optimization, codon adaptation index (CAI) of the gene raised from 0.50 to 0.99 and its GC content changed from 56 to 58. Expression analysis confirmed the presence of a protein band at 40 kDa. Furthermore, reticulocyte experiment results revealed that the activity of expressed darbepoetin alfa was similar to that of its equivalent expressed in Chinese hamster ovary (CHO) cells. These data suggested that the codon optimization and expression in L. tarentolae host provided an efficient approach for high level expression of darbepoetin alfa. © 2015 Elsevier Ltd. All rights reserved

Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae

Darbepoetin alfa is a biopharmaceutical glycoprotein that stimulates erythropoiesis and is used to treat anemia, which associated with renal failure and cancer chemotherapy. We herein describe the structural characterization of recombinant darbepoetin alfa produced by Leishmania tarentolae T7-TR host. The DNA expression cassette was integrated into the L. tarentolae genome through homologous recombination. Transformed clones were selected by antibiotic resistance, diagnostic PCRs, and protein expression analysis. The structure of recombinant darbepoetin alfa was analyzed by isoelectric focusing, ultraviolet–visible spectrum, and circular dichroism (CD) spectroscopy. Expression analysis showed the presence of a protein band at 40 kDa, and its expression level was 51.2 mg/ml of culture medium. Darbepoetin alfa have 5 isoforms with varying degree of sialylation. The UV absorption and CD spectra were analogous to original drug (Aranesp), which confirmed that the produced protein was darbepoetin alfa. Potency test results revealed that the purified protein was biologically active. In brief, the structural and biological characteristics of expressed darbepoetin alfa were very similar to Aranesp which has been normally expressed in CHO. Our data also suggest that produced protein has potential to be developed for clinical use. © 2016, Springer Science+Business Media New York

Expression of recombinant human insulin-like growth factor type 1 (rhiGF-1) in Escherichia Coli

Background: 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. Methods: 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. Results: 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. Conclusion: hIGF-1 expression cassette for cloning and expression in E. coli was designed 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. © 2015, Avicenna Journal of Medical Biotechnology. All rights reserved

Proline dehydrogenase (PRODH) gene polymorphisms and the risk of schizophrenia in Iranian populations

Schizophrenia is a highly heritable mental disorder which can be occurred as a result of mutations or single nucleotide polymorphisms (SNPs) in various genes. Proline dehydrogenase (PRODH) gene is one of the most important genes which can be associated with increased risk of schizophrenia in several populations. Here, we considered the effect of PRODH gene polymorphisms on the incidence of schizophrenia in Iranian populations. This study was done using the analysis of 3 SNPs markers, including G1496A, G758A and C1482T. Molecular analysis was performed on 263 schizophrenic patients and 278 healthy individuals (control group). These examinations were executed by PCR-based restriction fragment length polymorphism (RFLP) technique. Statistical analysis was performed by SPSS software (16.0). Our findings showed that G1496A and C1482T polymorphisms in patients were significantly higher than controls and there were meaningful correlations between the occurrence of these polymorphisms and schizophrenia in the population (P < 0.001). However­, there was no significant relationship between G758A in the PRODH gene and schizophrenia. Haplotype analysis showed that AAT, AAC and GAT blocks (variation alleles are bold) had significant correlations with schizophrenia. PRODH gene can be considered as one of the important genes involved in schizophrenia development among the Iranian population

Dose-dependent expression of claudin-5 is a modifying factor in schizophrenia

Schizophrenia is a neurodevelopmental disorder that affects up to 1% of the general population. Various genes show associations with schizophrenia and a very weak nominal association with the tight junction protein, claudin-5, has previously been identified. Claudin-5 is expressed in endothelial cells forming part of the blood-brain barrier (BBB). Furthermore, schizophrenia occurs in 30% of individuals with 22q11 deletion syndrome (22q11DS), a population who are haploinsufficient for the claudin-5 gene. Here, we show that a variant in the claudin-5 gene is weakly associated with schizophrenia in 22q11DS, leading to 75% less claudin-5 being expressed in endothelial cells. We also show that targeted adeno-associated virus-mediated suppression of claudin-5 in the mouse brain results in localized BBB disruption and behavioural changes. Using an inducible ‘knockdown’ mouse model, we further link claudin-5 suppression with psychosis through a distinct behavioural phenotype showing impairments in learning and memory, anxiety-like behaviour and sensorimotor gating. In addition, these animals develop seizures and die after 3–4 weeks of claudin-5 suppression, reinforcing the crucial role of claudin-5 in normal neurological function. Finally, we show that anti-psychotic medications dose-dependently increase claudin-5 expression in vitro and in vivo while aberrant, discontinuous expression of claudin−5 in the brains of schizophrenic patients post mortem was observed compared to age-matched controls. Together, these data suggest that BBB disruption may be a modifying factor in the development of schizophrenia and that drugs directly targeting the BBB may offer new therapeutic opportunities for treating this disorder

Cloning and expression of human recombinant erythropoietin in Leishmania tarentolae

Background and Objective: Human erythropoietin (EPO) is a glycosylated hormone with molecular weight of about 40 KDa which is synthesized in kidneys and plays an important role in proliferation and differentiation of erythrocytes.This study was done to assess and analyze the expression of recombinant EPO in Leishmania tarentolae host. Method: In this descriptive study, the EPO gene was codoned , optimized with bioinformatics database prior to be synthesized. It was cleaved by KpnIandXbaI enzymes and cloned into pLEXSY expression vector. The constructed expression cassette was transfected into Leishmania tarentolae through electroporaton method. Identification and confirmation of transfected colonies was performed using PCR expression diagnostic primers and EPO specific primers. Induction of the cloned gene was done with tetracycline. The expression in induced strains was analyzed by SDS-PAGE and western blotting techniques. The amount of recombinant protein was quantified by ELISA method. Confirmation of cloning and EPO expression cassette was carried out through genetic engineering procedures. Results: Expression analysis of transfected parasitic strain with SDS-PAGE and western blotting confirmed gene integration into chromosomal of host as well as expression. The optimal conditions for expression were found to be 10μg of tetracycline and 72h induction time. Molecular weight of expressed protein estimated to be 40 KDa and expression level was determined to be 12.4 mg/l which was equal to 1% of total protein mass. Conclusion: EPO expression cassette for cloning and expression in Leishmania tarentolae was designed and protein of interest was successfully induced and identified Leishmania tarentolae can be used as a suitable host for production of recombinant EPO and this technology has a potential for localization