Rational development of a polycistronic plasmid with a CpG-free bacterial backbone as a potential tool for direct reprogramming

Objective: Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector

Materials and Methods: In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in the E. coli GT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells

Results: In the present study, we developed a nonviral episomal vector named pLENSO/Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming

Conclusion: According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future

Expression and Activity Evaluation of Reteplase in Escherichia coli TOP10

Reteplase is a part of tissue plasminogen activator [t-PA] used for the removal of thrombi in blood vessels. In the present study we express the Reteplase gene in Escherichia coli TOP10 and then its thrombolytic activity was measured. The recombinant plasmid pBADgIIIA was transformed into the competent Escherichia coli TOP10 and then transformed bacteria was seeded into bioreactor containing 1.5 L LB medium and induced by 0.02% L-Arabinoseat 37 degreeC, pH 7, and 180 rpm until OD 600 of 0.6 was reached. Samples were analyzed by SDS-PAGE and western blotting and the expression of Reteplase was examined. Finally the activity of this recombinant protein was evaluated using Chromogenic Activity Assay Kit. The presence of Reteplase in transformed Escherichia coli TOP10 was examined by western blotting which revealed that the target protein in form inclusion body was expressed as a unique band at 39 and the refolded Reteplase was 66 KDa. The amount of protein produced was 90.5 microg/mL and its activity was determined as 0.8 units. In this study, the expression of Reteplase in Escherichia coli TOP10 was scaled up under optimum condition. Furthermore we earned Reteplase with partially suitable thrombolytic activity

Inactivation of aprE gene in bacillus subtilis 168 by homologus recombination

One of the most important producers of high quality industrial enzymes is the Gram-positive bacterium, Bacillus subtilis [B. Subtilis]. One major limitation that hinders the wide application of B. subtilis is the secretion of high levels of extracellular proteases which degrade the secreted foreign proteins. In this study, homologus recombination technique was used to knock out its protease gene, aprE. The internal segment of the pro-sequence of aprE gene of B. subtilis 168 with a length of 80 bps and its complementary sequence were synthesized and ligated into pUB110 at EcoR1 and XbaI restriction sites. Competent cells of B. subtilis 168 were prepared and transformed by electroporation using Bio Rad gene pulser as explained in the methods section. Transformants carrying the recombinant plasmid were selected for resistance to neomycin. The success of homologous recombination was checked by PCR amplification of the neomycin gene which was part of the vector and did not exist in the genome of B. subtilis 168. The protease activity was measured using the Protease Fluorescent Detection Kit based on the proteolytic hydrolysis of fluorescein isothiocyanate [FITC]-labeled casein-substrate. The results demonstrated that aprE gene would not be able to produce further active subtilisin E. The reduction of protease activity also confirmed the efficacy of the induced mutation in this gene. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis which limit the production of high quality protease- sensitive products such as lipase

Molecular cloning and expression of rat micro -opioid receptor in Escherichia coli [BL21]

The micro [mu] opioid receptors, which mediate the effects of morphine, are widely distributed in brain. The purpose of this study was to design a simple expression system for rat micro -receptor in Escherichia coli [BL21]. In this laboratory study, rat micro -receptor cDNA was isolated from pcDNA3 vector using Xba1 and Hind3 restriction enzymes. pET-15b was digested by Nco1 restriction enzyme. micro -receptor cDNA and pET-15b formed a recombinant DNA that was transformed to Escherichia coli [BL21]. The insert presence was proved by Rsa1 restriction enzyme and the induction of its expression was performed using IPTG. Finally, the presence of desired insert was confirmed using RSA1, and the colonies that had correct orientation in gene containing plasmid were used for further studies. On the SDS-page gel electrophoresis, a 33 kDa band was observed when IPTG was used at 0.5 and 1 mM concentrations, that is equal to calculated molecular weight of rat micro -receptor. At the end of this project, the expression of rat micro -receptor by IPTG induction was successfully performed

Cloning and expression of functional reteplase in escherichia coli TOP10

Production of tissue Plasminogen Activator protein [t-PA] in prokaryotes systems has many problems such as the lack of active protein production, multiple purification steps, and renaturation process which has been shown to be costly and time-consuming. In this study, reteplase which is the nonglycosylated active domain of t-PA was used to transform TOP10 Escherichia coli [E. coli] bacteria to resolve some of the above mentioned problems. Reteplase cDNA was ligated into pBAD/gIII plasmid which allowed secretion of this protein into the periplasmic space and would allow the correct formation of disulfide bonds in protein structure. The presence of reteplase cDNA in pBAD/gIII plasmid was confirmed by restriction digestion and sequencing. After induction of the expression of this protein by adding 0.0002% L-Arabinose to the medium, the proteins in periplasmic space as well as the inclusion bodies formed inside the cell were extracted. Subsequently, these proteins were purified and detected by Western blot method. Our results showed that the amount of reteplase extracted from periplasmic space was much lower than the extracted inclusion bodies and large quantities of the recombinant protein were present as inclusion bodies. Therefore, it was more efficient to use inclusion body extraction method for protein isolation and purification. We produced active reteplase after its expression in E. coli TOP10 and isolation of inclusion bodies produced the best results for purification and extraction of this protein

Optimization of the expression of genes encoding poly [3-hydroxyalkanoate] synthase from pseudomonas aeruginosa PTCC 1310 in escherichia coli

Over the years, the use of plastics has complicated the problem of disposal of solid wastes. One strategy to reduce plastic waste is the use of biodegradable plastics. A group of these plastics are polyhydroxyalkanoates [PHAs]. To date more than 250 different microorganisms are known to synthesize and accumulate PHA. Most Pseudomonas strains are able to accumulate mcl-PHA. In previous studies, the phaC1 and phaC2 genes were identified in Pseudomonas aeruginosa [P.aeruginosa] PTCC 1310 and were cloned. The aim of this study was to express these genes and optimize the conditions for their expression. The inserts obtained from vectors pTZPHAC1 and pTZPHAC2 were subcloned into pET15b expression vector. After transformation of competent Escherichia coli [E.coli] BL21 [DE3] cells with recombinant plasmids, expression was induced using IPTG. By changing expression conditions such as IPTG concentration, time and temperature of incubation with IPTG, the expression conditions for these enzymes were optimized, and the obtained results were compared using proper statistical analysis. The PHA synthase genes were induced with IPTG and the expressed 62 kDa protein was observed and purified. By changing expression conditions, 1 mM IPTG, 37°C and a 2 hr incubation provided the highest level of protein production in E.coli cells. These results suggest that induction condition of PhaC genes can influence expression of PHA synthase enzymes