Cloning, high-level gene expression and bioinformatics analysis of SP15 and LeIF from Leishmania major and Iranian Phlebotomus papatasi saliva as single and novel fusion proteins: a potential vaccine candidate against leishmaniasis.

BACKGROUND: Early exacerbation of cutaneous leishmaniasis is mainly affected by both the salivary and Leishmania parasite components. Little is known of the vaccine combination made by immunogenic proteins of sandfly saliva (SP15) with Leishmania parasites (LeIF) as a single prophylactic vaccine, namely SaLeish. Also, there are no data available to determine the species-specific sequence of SP15 isolated from the Iranian Phlebotomus papatasi. METHODS: Integrated bioinformatics and genetic engineering methods were employed to design, optimize and obtain a vector-parasite-based vaccine formulation in a whole-length fusion form of LeIF-SP15 against leishmaniasis. Holistic gene optimization was initially performed to obtain a high yield of pure 'whole-SaLeish' expression using bioinformatics analyses. Genomic and salivary gland RNAs of wild-caught P. papatasi were extracted and their complementary DNA was amplified and cloned into pJET vector. RESULTS: The new chimeric protein of whole-SaLeish and randomly selected transcripts of native PpIRSP15 (GenBank accession nos. MT025054 and MN938854, MN938855 and MN938856) were successfully expressed, purified and validated by immunoblotting assay. Furthermore, despite the single amino acid polymorphisms of PpIRSP15 found at positions Y23 and E73 within the population of wild Iranian sandflies, antigenicity and conservancy of PpIRSP15 epitopes remained constant to activate T cells. CONCLUSIONS: The SaLeish vaccine strategy takes advantage of a plethora of vector-parasite immunogenic proteins with potential protective efficacy to stimulate both the innate and specific cellular immune responses against Leishmania parasites.

Response surface methodology to optimize partition and purification of two recombinant oxidoreductase enzymes, glucose dehydrogenase and d-galactose dehydrogenase in aqueous two-phase systems.

Oxidoreductases are an important family of enzymes that are used in many biotechnological processes. An experimental design was applied to optimize partition and purification of two recombinant oxidoreductases, glucose dehydrogenase (GDH) from Bacillus subtilis and d-galactose dehydrogenase (GalDH) from Pseudomonas fluorescens AK92 in aqueous two-phase systems (ATPS). Response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to optimize critical factors like polyethylene glycol (PEG) concentration, concentration of salt and pH value. The best partitioning conditions was achieved in an ATPS composed of 12% PEG-6000, 15% K2HPO4 with pH 7.5 at 25°C, which ensured partition coefficient (KE) of 66.6 and 45.7 for GDH and GalDH, respectively. Under these experimental conditions, the activity of GDH and GalDH was 569.5U/ml and 673.7U/ml, respectively. It was found that these enzymes preferentially partitioned into the top PEG-rich phase and appeared as single bands on SDS-PAGE gel. Meanwhile the validity of the response model was confirmed by a good agreement between predicted and experimental results. Collectively, according to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of any enzyme from oxidoreductase family.

Polymorphism of the CLDN5 gene and Schizophrenia in an Iranian Population.

BACKGROUND: The gene coding claudin (CLDN5) is located on 22q11. Since the proteins of CLDN5 family are a ma-jor component for barrier-forming tight junctions, it may be important to test whether or not the CLDN5 locus could be associated with schizophrenia. METHOD: A total of 150 individuals affected with schizophrenia and 150 healthy persons were recruited. The relation-ship between the three single nucleotide polymorphism (SNPs) and schizophrenia disease was studied using polymer-ase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) technique. The PCR products were completely digested with restriction enzymes of DpnII, PvuII and BstNI, and then separated on agarose gel. The statis-tical investigations and haplotype analysis were also performed. RESULTS: The transmission disequilibrium test (TDT) exhibited weak association between rs10314 [C/G] and schizo-phrenia (v2 = 3.55, P = 0.022), but the other two SNPs did not show such an association. The global chi-square test showed that the 3-SNP haplotype system was not associated with schizophrenia although the 1-df test for individual haplotypes showed that the rs1548359(C)-rs10314(G)-rs739371(C) haplotype was excessively non-transmitted (v2 = 6.33, P = 0.025). The v2 test for LD between SNPs indicated that these three SNPs were in strong LD. CONCLUSIONS: Collectively, LD analysis showed that the CLDN5 locus was associated with schizophrenia in an Iranian population.

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.