Cloning, expression and purification of pseudomonas putida ATCC12633 Creatinase

Background: Pseudomonas putida [P. putida] ATCC12633 can produce creatinase. It is a microbial enzyme which degrades creatinine in bacteria and provides source of carbon and nitrogen. Also, this enzyme is used in the enzymatic measurement of creatinine concentration for diagnosis of renal and muscles functions and diseases. Our purpose was recombinant production of creatinase for using in clinical measurement of serum or urine creatinine

Methods: A 1209bp of open reading frame of creatinase was amplified by PCR from P. putida ATCC12633 genome and cloned into pET[2]8a expression vector which was digested using NheI and XhoI restriction enzymes. Cloning was confirmed by colony PCR, double digestion analysis and sequencing. Recombinant pET[2]8a vector was transformed to Escherichia coli [E. coli] BL21 [DE3]. Creatinase expression was induced in E.coli BL21 [DE3] using IPTG and confirmed by SDS-PAGE and western blotting. Purification of creatinase was performed using Ni-NTA column. The specific activity of this enzyme was also investigated

Results: The creatinase gene cloning was confirmed by DNA sequencing. Successful expression of creatinase was performed in E. coli [57.4% of total protein]. SDS-PAGE and western blot analysis showed a 45 kDa creatinase protein. Purification of creatinase was done with high purity. The specific activity of recombinant enzyme is 26.54 unit/mg that is much higher than other creatinase used in the commercial kits [9 unit/mg]

Conclusion: The P. putida ATCC12633 recombinant creatinase was expressed efficiently in E. coli BL21 and 57% of total protein was the recombinant creatinase. Also, expressed creatinase has high solubility and also the enzyme has good activity compared to enzymes used in commercial kits, so a new source of creatinase was produced for creatinine assay kit in this study

Differentiation of definitive endoderm from human induced pluripotent stem cells on hMSCs feeder in a defined medium

Background: The Definitive Endoderm [DE] differentiation using the undefined media and non-human feeders can cause contaminations in the generated cells for therapeutic applications. Therefore, generating safer and more appropriate DE cells is needed. This study compared five different methods to establish an appropriate method for inducing an efficient DE differentiation from Human Induced Pluripotent Stem Cells [hiPSCs] on an appropriate feeder in a more defined medium

Methods: Human Induced Pluripotent Stem Cells [hiPSCs] were cultured on inactivated feeders. Passaged hiPSCs, without feeder, were incubated for three days with Activin-A and different endodermal differentiation media including 1-FBS, 2-B27, 3- ITS and albumin fraction-V, 4-B27 and ITS and 5-like the third medium. The feeder cells in the first four methods were Mouse Embryonic Fibroblasts [MEFs] and in the fifth method were human adult bone marrow Mesenchymal Stem Cells [hMSCs]. DE markers FOXA2, SOX17 and CXCR4 and also pluripotency marker OCT4 were evaluated using qRT-PCR, as well as FOXA2 by the immunocytochemistry

Results: QRT-PCR analysis showed that after three days, the expression levels of DE and pluripotency markers in the differentiated hiPSCs among all five groups did not have any significant differences. Similarly, the immunocytochemistry analysis demonstrated that the differentiated hiPSCs expressed FOXA2, with no significant differences

Conclusion: Despite this similarity in the results, the third differentiation medium has more defined and cost effective components. Furthermore, hMSC, a human feeder, is safer than MEF. Therefore, the fifth method is preferable among other DE differentiation methods and can serve as a fundamental method helping the development of regenerative medicine

Design and bioinformatics analysis of novel biomimetic peptides as nanocarriers for gene transfer

The introduction of nucleic acids into cells for therapeutic objectives is significantly hindered by the size and charge of these molecules and therefore requires efficient vectors that assist cellular uptake. For several years great efforts have been devoted to the study of development of recombinant vectors based on biological domains with potential applications in gene therapy. Such vectors have been synthesized in genetically engineered approach, resulting in biomacromolecules with new properties that are not present in nature. In this study, we have designed new peptides using homology modeling with the purpose of overcoming the cell barriers for successful gene delivery through Bioinformatics tools. Three different carriers were designed and one of those with better score through Bioinformatics tools was cloned, expressed and its affinity for pDNA was monitored. The resultszz demonstrated that the vector can effectively condense pDNAinto nanoparticles with the average sizes about 100 nm. We hope these peptides can overcome the biological barriers associated with gene transfer, and mediate efficient gene delivery

Evaluation of cell penetrating peptide delivery system on HPV16E7 expression in three types of cell line

Background: the poor permeability of the plasma and nuclear membranes to DNA plasmids are two major barriers for the development of these therapeutic molecules. Therefore, success in gene therapy approaches depends on the development of efficient and safe non-viral delivery systems

Objectives: the aim of this study was to investigate the in vitro delivery of plasmid DNA encoding HPV16 E7 gene using cell penetrating peptide delivery system to achieve the best conditions for cell transfection and protein expression. For this purpose, we have used a cationic peptide delivery system, MPG which forms stable non-covalent complexes with nucleic acids for delivery of pEGFP-E7 as a model antigen in vitro

Materials and Methods: DNA construct encoding HPV16 E7 [pEGFP-E7] was prepared in large scale with high purity. MPG peptide/ DNA complexes were prepared at different N/P [nitrogen/phosphate] ratios and physicochemical characterization and stability of nanoparticles were investigated. In vitro peptide-mediated E7-GFP DNA transfection, and its expression was evaluated in three cell types. To quantify the transfection efficiency of this delivery system, transfected cells were harvested and assessed for GFP-positive cells by flow cytometry. Furthermore, E7-GFP expression was confirmed by western blot analysis

Results: the cellular uptake of MPG based nanoparticles was shown to be comparable with standard reagent PEI. The COS-7 cells transfected by MPG-based nanoparticles at an N/P ratio of 15:1 showed the highest transfection efficiency and gene expression

Conclusions: the results indicated that the efficient gene expression depends on both cell type and N/P ratio applied, in vitro. The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPGbased nanoparticles as a potent gene delivery system

comparative study of osteogenic differentiation human induced pluripotent stem cells and adipose tissue derived mesenchymal stem cells

Human induced pluripotent stem cells [iPSCs] have been shown to have promising capacity for stem cell therapy and tissue engineering applications. Therefore, it is essential to compare the ability of these cells with the commonly used mesenchymal stem cells [MSC] for bone tissue engineering in vitro. In this experimental study, the biological behavior and osteogenic capacity of the iPSCs were compared with MSCs isolated from human adipose tissue [AT-MSCs] using 3-[4,5-di-methylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT] assay, Alizarin red staining, alkaline phosphatase [ALP] activity measurements, calcium content assay and common osteogenic-related genes. Data were reported as the mean +/- SD. One-way analysis of variance [ANOVA] was used to compare the results. A p value of less than 0.05 was considered statistically significant. There was a significant difference between the rate of proliferation of the two types of stem cells; iPSCs showed increased proliferation compared to AT-MSCs. During osteogenic differentiation, ALP activity and mineralization were demonstrated to be significantly higher in iPSCs. Although AT-MSCs expressed higher levels of Runx2, iPSCs expressed higher levels of osteonection and osteocalcin during differentiation. iPSCs showed a higher capacity for osteogenic differentiation and hold promising potential for bone tissue engineering and cell therapy applications

effect of vitrification and in vitro culture on the adenosine triphosphate content and mitochondrial distribution of mouse pre-implantation embryos

The mitochondria are an important source of adenosine triphosphate [ATP] production in pre-implantation embryo. Therefore, the objective of this study was to investigate the effect of vitrification and in vitro culture of mouse embryos on their mitochondrial distribution and ATP content. The embryos at 2-PN, 4-cell and blastocyst stages were collected from the oviduct of stimulated pregnant mice and uterine horns. Then, the embryos were vitrified with the cryotop method using ethylene glycol and dimethylsulphoxide. After evaluating the survival rates of vitrified embryos, their development to hatching stages were assessed. The ATP content of collected in vivo and in vitro embryos at different stages was measured by luciferin-luciferase bioluminescence assay. The distribution of mitochondria was studied using Mito-tracker green staining under a fluorescent microscope. The survival rates of vitrified embryos at 2-PN, 4-cell and early blastocyst stages were 84.3, 87.87 and 89.89%, respectively. The hatching rates in previous developmental stages in vitrified group were 57.44, 66.73 and 70.89% and in non-vitrified group were 66.32, 73.25 and 75.89%, respectively [P>0.05] The ATP content of in vivo or in vitro collected embryos was not significantly different in both vitrified and non-vitrified groups [P>0.05]. Mitochondrial distribution of vitrified and non-vitrified 2-PN embryos was similar, but some clampings or large aggregation of mitochondria within the vitrified 4-cell embryos was prominent. Vitrification method did not affect the mouse embryo ATP content. Also, the cellular stress was not induced by this procedure and the safety of vitrification was shown

[Comparison of survival and developmental rates of mouse ovarian follicles after two and three dimensional cultures]

In vitro ovarian follicle culture provides a tool to investigate folliculogenesis and may be used as an assisted reproductive technology [ART]. This study aims to compare survival and development rates in mouse ovarian follicles after two and three dimensional in vitro cultures. Preantral follicles were isolated from the ovaries of 14-day old female mice and cultured in alpha-MEM medium supplemented with 5% FBS for 12 days in a two dimensional and three dimensional culture with different concentrations of sodium alginate [0.25%, 0. 5%, or 1%]. The follicle diameter, survival and maturation rate during culture were analyzed and compared with one-way analysis of variance [ANOVA]. P<0.05 was considered as statistically significant. The mean diameter of preantral follicles that capsulated with 0.5% alginate was significantly higher than other concentrations in each group on days 6 and 12 [P<0.001]. The percentages of follicles which released metaphase two [MII] oocytes in the two dimensional groups and in the three dimensional groups at 0.25%, 0.5% and 1% concentrations of sodium alginate were 29.03%, 33.33%, 44.18% and 35.89% respectively. The percentage of MII oocytes was significantly higher at the 0.5% concentration of sodium alginate [P<0.001]. Follicles encapsulated in 0. 5% sodium alginate in three dimensional culture displayed the highest survival development and maturation rate.

[ effects of p-benzoquinone and hydroquinone on the RUNX2 gene expression and osteoblastic differentiation of human marrow derived mesenchymal stem cells]

Evaluating the effects of p-benzoquinone and hydroquinone on the RUNX2 expression and osteoblastic differentiation of human marrow derived mesenchymal stem cells [MSCs]. Bone marrow MSCs obtained by cultivating marrow mononuclear cells, were exposed to 10?M of either p-benzoquinone or hydroquinone. Following chemical treatment, RUNX2 gene expression was assessed by Real-time RT PCR 1, 6, 24 and 48 hours later and osteogenic differentiation was analyzed using alizarin red and alkaline phosphatase staining methods on days 7 and 14 after ostegenic induction. RUNX2 expression was significantly elevated [up to approximately 8 times] due to chemical exposure but the applied chemicals exert no considerable effect on MSCs osteogenic differentiation. According to the literature, despite the necessity of RUNX2 overexpression on the induction of osteogenic differentiation, but it is not sufficient for osteogenesis to occure so increase in RUNX2 expression observed in our study is not the indicator of the induced osteogenic differentiation. Instead, this elevated expression could be the sign of increased activity of the canonical Wnt signaling pathway thereby its involvement in the development of AML due to exposure to benzene and its metabolites. Moreover, this augmented expression of RUNX2 in MSCs can indicate the RUNX2 overexpression in myeloid progenitors as an expected similar effect of exposure to benzene and its metabolites to contribute in myeloid malignancies developed due to benzene exposure

[Bacterial overexpression of the human interleukin-2 in insoluble form via the pET Trx fusion system]

Selection of a system for successful recombinant protein production is important. The aim of this study was to produce high levels of human interleukin-2 [hIL-2] in soluble form. To this end, the pET32a vector in Escherichia coli BL21 [DE3] was used as an expression system, since it was previously used for the production of mouse IL-2 in soluble form. The results indicated that contrary to expectations, the expressed protein was in the form of inclusion bodies and perhaps amino acid differences between human and mouse IL-2 should be determinant. The hIL-2 protein is a small peptide, therefore its recovery as a biologically functional protein by the process of refolding may be feasible and could lead to high yields at the industrial scale