Rational design of a new variant of Reteplase with optimized physicochemical profile and large-scale production in Escherichia coli.

Structural engineering of the recombinant thrombolytic drug, Reteplase, and its cost-effective production are important goals in the pharmaceutical industry. In this study, a single-point mutant of the protein was rationally designed and evaluated in terms of physicochemical characteristics, enzymatic activity, as well as large-scale production settings. An accurate homology model of Reteplase was used as the input to appropriate tools to identify the aggregation-prone sites, while considering the structural stability. Selected variants underwent extensive molecular dynamic simulations (total 540 ns) to assess their solvation profile and their thermal stability. The Reteplase-fibrin interaction was investigated by docking. The best variant was expressed in E. coli, and Box-Behnken design was used through response surface methodology to optimize its expression conditions. M72R mutant demonstrated appropriate stability, enhanced enzymatic activity (p < 0.05), and strengthened binding to fibrin, compared to the wild type. The optimal conditions for the variant's production in a bioreactor was shown to be 37 ºC, induction with 0.5 mM IPTG, for 2 h of incubation. Under these conditions, the final amount of the produced enzyme was increased by about 23 mg/L compared to the wild type, with an increase in the enzymatic activity by about 2 IU/mL. This study thus offered a new Reteplase variant with nearly all favorable properties, except solubility. The impact of temperature and incubation time on its large-scale production were underlined as well.

Optimization of solvent media to solubilize TEV protease using response surface method.

BACKGROUND AND PURPOSE: Tobacco etch virus (TEV) protease, a 27 KDa protein, consists of the catalytic domain of nuclear inclusion a (NIa) protein produced by Tobacco etch virus. Because of its unique sequence, TEV protease is used for purging fusion tags from proteins. It also has many advantages such as stability and activity in a board range of temperature and pH and overproduction in Escherichia coli and these benefits make this protease valuable. Despite all these benefits, TEV protease has problems like low solubility (less than 1 mg/mL). There are methods for enhancing protein solubility and in this study, the effect of additives during cell lysis was studied. EXPERIMENTAL APPROACH: Eleven different additives that made twelve different lysis buffers were used and their effect on TEV protease solubility analyzed by Plackett-Burman and response surface methodology methods. FINDINGS / RESULTS: Three best effective additives on TEV solubility (L-proline, sodium selenite, and CuCl2) were selected according to software analysis and the best concentration of them was applied to optimize TEV protease solubility. CONCLUSION AND IMPLICATIONS: The obtained results provided the composition of an optimum solvent for obtaining soluble TEV protease.

Investigation of Supercharging as A Strategy to Enhance the Solubility and Plasminogen Cleavage Activity of Reteplase.

BACKGROUND: Reteplase, the recombinant form of tissue plasminogen activator, is a thrombolytic drug with outstanding characteristics, while demonstrating limited solubility and reduced plasminogen activation. Previously, we in silico designed a variant of Reteplase with positively supercharged surface, which showed promising stability, solubility and activity. This study was devoted to evaluation of the utility of supercharging technique for enhancing these characteristics in Reteplase. OBJECTIVE: To test the hypothesis that reinforced surface charge of a rationally-designed Reteplase variant will not compromise its stability, will increase its solubility, and will enhance its plasminogen cleavage activity. MATERIALS AND METHODS: Supercharged Reteplase coding sequence was cloned in pDest527 vector and expressed in E. coli BL21 (DE3). The expressed protein was extracted by cell disruption. Inclusion bodies were solubilized using guanidine hydrochloride, followed by dialysis for protein refolding. After confirmation with SDS-PAGE and western blotting, extracted proteins were assayed for solubility and tested for bioactivity. RESULTS: SDS-PAGE and western blot analysis confirmed the successful expression of Reteplase. Western blot experiments showed most of Reteplase expressed in the insoluble form. Plasminogen cleavage assay showed significantly higher activity of the supercharged variant than the wild type protein (P < 0.001). The stability of the supercharged variant was also comparable to the wild type. CONCLUSION: Our findings, i.e. the contribution of the surface supercharging technique to retained stability, enhanced plasminogen cleavage activity, while inefficiently changed solubility of Reteplase, contain implications for future designs of soluble variants of this fibrinolytic protein drug.

Improvement of Soluble Production of Reteplase in Escherichia coli by Optimization of Chemical Chaperones in Lysis Buffer.

BACKGROUND: Reteplase is a nonglycosylated derivative of recombinant tissue plasminogen activator, a thrombolytic agent, which can be easily expressed in Escherichia coli. However, overexpression of reteplase in E. coli usually leads to accumulation of insoluble and inactive aggregates and inclusion bodies. In the present study, we aimed to optimize chemical additives of lysis buffer to avoid the initial aggregation and formation of inclusion bodies of reteplase at cell disruption step. MATERIALS AND METHODS: After protein expression in E. coli BL21 (DE3), the bacterial cells were disrupted in different lysis buffers using microsmashing. Eleven chemical additives at two concentration levels were combined based on a Plackett-Burman design to prepare 12 different lysis buffers used at cell disruption stage. Then, three additives with the most positive effect on improvement of solubility of reteplase were chosen and used for the second screening based on Box-Behnken model. RESULTS: The primary screening results showed that among 11 additives, arginine, K2PO4, and cetyltrimethylammonium bromide (CTAB) had the most positive effect on solubility of reteplase. Our final results based on 14 runs of Box-Behnken design showed that the optimum buffer additive condition is 0.005 mg/ml CTAB, 0.065 mg/ml arginine, and 0.026 mg/ml K2PO4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Western blotting of soluble and total fraction of samples confirmed that these additives significantly improved soluble production of reteplase compared with control. CONCLUSION: Our study indicates that the application of chemical additives in cell lysis can improve the solubility of reteplase. Further studies are still required to understand the exact mechanism of chemical additives as a chemical chaperone during cell lysis.

Improving the solubility, activity, and stability of reteplase using in silico design of new variants.

Reteplase (recombinant plasminogen activator, r-PA) is a thrombolytic agent recombined from tissue-type plasminogen activator (t-PA), which has several prominent features such as strong thrombolytic ability and E. coli expressibility. Despite these outstanding features, it demonstrates reduced fibrin binding affinity, reduced stimulation of protease activity, and lower solubility, hence higher aggregation propensity, compared to t-PA. The present study was devoted to design r-PA variants with comparable structural stability, enhanced biological activity, and high solubility. For this purpose, computational molecular modeling techniques were utilized. The supercharging technique was applied for r-PA to designing new species of the protein. Based on the results from in silico evaluation of selected mutations in comparison to the wild-type r-PA, the designed supercharged mutant (S7 variant) exhibited augmented stability, decreased solvation energy, as well as enhanced binding affinity to fibrin. The data also implied increased plasminogen cleavage activity of the new variant. These findings have implications to therapies which involve removal of intravascular blood clots, including the treatment of acute myocardial infarction.

Optimization of Fermentation Conditions for Reteplase Expression by Escherichia coli Using Response Surface Methodology.

BACKGROUND: Expression of heterologous proteins at large scale is often a challenging job due to plasmid instability, accumulation of acetate and oxidative damage in bioreactors. Therefore, it is necessary to optimize parameters influencing cell growth and expression of recombinant protein. METHODS: In the present study, the optimal culture conditions for expression of reteplase by Escherichia coli (E. coli) BL21 (DE3) in a bench-top bioreactor was determined. Response Surface Methodology (RSM) based on Box-Behnken design was used to evaluate the effect of three variables (i.e., temperature, shaking speed and pH) and their interactions with cellular growth and protein production. The obtained data were analyzed by Design Expert software. RESULTS: Based on results of 15 experiments, a response surface quadratic model was developed which was used to explain the relation between production of reteplase and three investigated variables. The high value of "R-Squared" (0.9894) and F-value of 51.99 confirmed the accuracy of this model. According to the developed model, the optimum fermentation conditions for reteplase expression were temperature of 32°C, shaking speed of 210 rpm, and pH of 8.4. This predicted condition was applied for the production of reteplase in the bioreactor leading to a protein yield of 188 mg/l. CONCLUSION: Our results indicate the significant role of culture conditions (e.g., pH, temperature and oxygen supply) in protein expression at large scale and confirm the need for optimization. The proposed strategy here can also be applied to experimental set-up of optimization for fermentation of other proteins.

Reteplase: Structure, Function, and Production.

Thrombolytic drugs activate plasminogen which creates a cleaved form called plasmin, a proteolytic enzyme that breaks the crosslinks between fibrin molecules. The crosslinks create blood clots, so reteplase dissolves blood clots. Tissue plasminogen activator (tPA) is a well-known thrombolytic drug and is fibrin specific. Reteplase is a modified nonglycosylated recombinant form of tPA used to dissolve intracoronary emboli, lysis of acute pulmonary emboli, and handling of myocardial infarction. This protein contains kringle-2 and serine protease domains. The lack of glycosylation means that a prokaryotic system can be used to express reteplase. Therefore, the production of reteplase is more affordable than that of tPA. Different methods have been proposed to improve the production of reteplase. This article reviews the structure and function of reteplase as well as the methods used to produce it.

Study of the Role of siRNA Mediated Promoter Methylation in DNMT3B Knockdown and Alteration of Promoter Methylation of CDH1, GSTP1 Genes in MDA-MB -453 Cell Line.

Promoter methylation is one of the main epigenetic mechanisms that leads to the inactivation of tumor suppressor genes during carcinogenesis. Due to the reversible nature of DNA methylation, many studies have been performed to correct theses epigenetic defects by inhibiting DNA methyltransferases (DNMTs). In this case novel therapeutics especially siRNA oligonucleotides have been used to specifically knock down the DNMTs at mRNA level. Also many studies have focused on transcriptional gene silencing in mammalian cells via siRNA mediated promoter methylation. The present study was designed to assess the role of siRNA mediated promoter methylation in DNMT3B knockdown and alteration of promoter methylation of Cadherin-1 (CDH1), Glutathione S-Transferase Pi 1(GSTP1), and DNMT3B genes in MDA-MB-453 cell line. MDA-MB-453 cells were transfected with siDNMT targeting DNMT3B promoter and harvested at 24 and 48 h post transfection to monitor gene silencing and promoter methylation respectively. DNMT3B expression was monitored by quantitative RT-PCR method. Promoter methylation was quantitatively evaluated using differential high resolution melting analysis. A non-significant 20% reduction in DNMT3B mRNA level was shown only after first transfection with siDNMT, which was not reproducible. Promoter methylation levels of DNMT3B, CDH1, and GSTP1 were detected at about 15%, 70% and 10% respectively, in the MDA-MB-453 cell line, with no significant change after transfection. Our results indicated that siDNMT sequence were not able to affect promoter methylation and silencing of DNMT3B in MDA-MB-453 cells. However, quantitation of methylation confirmed a hypermethylated phenotype at CDH1 and GSTP1 promoters as well as a differential methylation pattern at DNMT3B promoter in breast cancer.

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.

Optimization of the Expression of Reteplase in Escherichia coli TOP10 Using Arabinose Promoter.

BACKGROUND: Reteplase is a mutant version of t-PA (tissue plasminogen activator) with prolonged half-life. In the present study, E. coli Top 10 bacteria were utilized in the production of reteplase, which is the nonglycosylated active domain of t-PA. Reteplase gene was ligated into pBAD/gIII plasmid which, allows secretion of this protein in periplasmic space. It would allow the correct formation of disulfide bonds in protein structure. OBJECTIVES: This study aimed at expression of reteplase in optimum condition. In this study, the reteplase gene was cloned and expressed in Escherichia coli top 10 as a suitable host cell and its expression was optimized. MATERIALS AND METHODS: The recombinant plasmid, pET15b/reteplase was digested by NcoI and BamHI restriction enzymes; while pBAD/gIIIA vector was digested by NcoI and BglII. Then the insert and vector were ligated and used for transformation of E. coli Top10 cells by heat shock method. Overnight culture of transformed bacteria was induced by L-arabinose in various concentrations (0.2, 0.02, 0.002, and 0.0002%) and at various temperatures. RESULTS: The obtained recombinant plasmid was sequenced to confirm the presence and correct framing of reteplase gene regarding the expression of reteplase. Maximum production of this enzyme was obtained under the following condition: 0.0002% L-arabinose at 37°C for 2 hours incubation. The purified protein was detected on SDS-PAGE (sodium dodecyl sulfate Polyacrylamide gel electrophoresis) as a 66 kDa band. The concentration of t-PA standard was 1 unit which is equal to 12 µg/mL. The enzymatic activity of samples was measured as 0.8 units compared to the standards. CONCLUSIONS: Reteplase was expressed in E. coli Top 10 after activation of pBAD/gIIIA promoter region by arabinose and optimized.

Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli.

The human epidermal growth factor receptor (HER) family plays an important role in cell growth and signaling and alteration of its function has been demonstrated in many different kinds of cancer. Receptor dimerization is necessary for the HER signal transduction pathway and tyrosine kinase activity. Recently, several monoclonal antibodies have been developed to directly interfere with ligand-HER receptor binding and receptor dimerization. A single chain variable fragment (ScFv) is a valuable alternative to an intact antibody. This report describes the production and purification of an ScFv specific for domain II of the HER2 receptor in Escherichia coli BL21 (DE3) cytoplasm. The majority of expressed of anti-her2his-ScFv protein was produced as inclusion bodies. A Ni-NTA affinity column was used to purify the anti-her2his-ScFv protein. The molecular weight of anti-her2his-ScFv protein was estimated to be approximately 27 kDa, as confirmed by SDS-PAGE and Western blotting assay. The anti-her2his-ScFv showed near 95 % purity and reached a yield of approximately 29 mg/l in flask fermentation. The purified anti-her2his-ScFv showed its biological activity by binding to HER2 receptor on the surface of BT-474 cells. This ScFv may be a potential pharmaceutical candidate for targeting tumour cells overexpressing HER2 receptor.