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.

High-yield Production of Granulocyte-macrophage Colony-stimulating Factor in E. coli BL21 (DE3) By an Auto-induction Strategy.

A novel strategy to increase protein expression yield is unintended induction of expression in complex media, called auto-induction. This method can be used to express proteins under control of the lac promoter without any need to monitor bacterial growth pattern, and addition of specific expression inducers such as Isopropyl ß-D-1-thiogalactopyranoside (IPTG) at proper time. In the present study, a codon optimized gene encoding granulocyte-macrophage colony stimulating factor (GM-CSF) was cloned and over-expressed in Escherichia coli BL21 (DE3) using both conventional inducer-based and auto-induction approaches in a shake flask scale and the yield of GM-CSF expression and biomass production was identified. Results showed higher biomass production and expression yield for GM-CSF in case of auto-induction comparing with IPTG-induction. The auto-induction approach was also performed in a fed batch fermentation process in a 2-L bioreactor scale. The feeding strategy yielded an amount of 300 mg/L GM-CSF within 20 h of induction. However, most of the over-expressed GM-CSF was produced as inclusion bodies and following purification and refolding, a final yield of 90 mg/L was achieved. These results suggest that auto-induction approach can be effectively applied in fed-batch fermentation for the large scale production of GM-CSF; however, further optimization of purification process is obligatory to increase the purification yield.

Improvement of soluble expression of GM-CSF in the cytoplasm of Escherichia coli using chemical and molecular chaperones.

The most common approaches to improve soluble expression of heterologous proteins are applications of molecular chaperones such as DnaK, DnaJ, GrpE, GroEL and GroES. The aim of present study was to enhance soluble expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in Escherichia coli by different approaches including modification of cultivation and induction conditions, and thermally, genetically and chemically enhancement of expression of cellular chaperones. To genetically enhance amount of molecular chaperones, co-expression of pET28-GM-CSF and pKJE7 plasmids was performed. The soluble expressed protein was affinity purified and subjected to endotoxin removal. Co-expression with molecular chaperones significantly increased soluble expression of GM-CSF. Addition of chemical chaperones and osmolytes like NaCl (0.5 M), sucrose (0.5 M), sorbitol (0.5 M) and MgCl2 (1 mM) to growing media could improve solubility of GM-CSF. Biological activity of purified GM-CSF was confirmed based on its proliferative effect on HL-60 cell lines. The approach developed in the present study can be applied to improve soluble expression of other recombinant protein proteins.

Cloning, Optimization of Periplasmic Expression and Purification of Recombinant Granulocyte Macrophage-Stimulating Factor in Escherichia coli BL21 (DE3).

BACKGROUND: Molgramostim, a nonglycosylated version of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), can be produced in a high level by Escherichia coli. However, overexpression of GM-CSF in bacterial cells usually leads to formation of inclusion bodies and insoluble protein aggregates which are not biologically active. The aim of the present study was to improve the expression of soluble and biologically active GM-CSF in periplasmic space of E. coli BL21 (DE3). MATERIALS AND METHODS: The codon-optimized GM-CSF gene was subcloned into pET-22b expression vector, in frame with the pelB secretion signal peptide for periplasmic secretion. Cultivation conditions including as isopropyl ß-D-1-thiogalactopyranoside (IPTG) concentration, incubation temperature, and presence of sucrose were optimized to improve periplasmic expression of GM-CSF. The expressed protein was purified using Ni-NTA affinity column. Biological activity of GM-CSF on HL-60 cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The amount of soluble protein for periplasmic expression was more when compared with one of the cytoplasmic expressions. The optimum condition for periplasmic expression of GM-CSF was expression at 23°C, using 1 mM IPTG as inducer and in the presence of 0.4 M sucrose. The biological activity of purified GM-CSF on HL-60 cell line was assessed by MTT assay, and the specific activity of produced GM-CSF was determined as 1.2 × 104 IU/µg. CONCLUSION: The present work suggests that periplasmic expression and optimization of cultivation conditions could improve soluble expression of recombinant proteins by E. coli.

Efficient Media for High Lipase Production: One Variable at a Time Approach.

BACKGROUND: Lipase enzymes have applications in a wide range of industries. A crucial determining factor of industrial prices of these enzymes is the culture media composition that is constantly under review by researchers. In this work, for maximum lipase production by Bacillus sp. ZR-5, culture media compositions were optimized using ″one variable at a time″ strategy. METHODS: For this purpose, the culture medium parameters such as low and high cost carbon and nitrogen sources, substrates and incubation times were evaluated. RESULTS: Maximum lipase activity was achieved after 24 hr of incubation with 1.5% of glucose syrup (1600±69.1 u/mg), 1% of fish powder (1238±36.7 u/mg) and olive oil (1407±2.1 u/mg) as low cost carbon and nitrogen sources and substrate, respectively. CONCLUSION: Our results show a significant increase in lipase activity with usage of low cost sources; this could help in reducing the media prices for industrial application of lipase enzyme.

Twin arginine translocation system in secretory expression of recombinant human growth hormone.

Recombinant protein production in E. coli has several advantages over other expression systems. Misfolding, inclusion body formation, and lack of eukaryotic post translational modification are the most disadvantages of this system. Exporting of correctly folded proteins to the outside of reductive cytoplasmic environment through twin-arginine system could help to pass these limiting steps. Two signal sequences, TorA and SufI are used at N-terminal of human growth hormone (hGH) bearing DsbA gene sequence at C-terminal to enhance folding. The synthetic cassettes including the signal sequence, hGH and DsbA were transformed into E. coli BL21 (DE3) to study the effect of signal sequence and DsbA chaperone on translocation and folding of the protein. The results confirmed using signal sequence at N-terminal of targeted protein and coexpression with DsbA could transport proteins to the periplasmic space and culture media compared to control groups. Although there is no protein band of somatropin in SDS-Page of culture media samples when using SufI as signaling sequence, the study demonstrated TorA signal sequence could transport the target protein to the culture media. However, there was a considerable amount of hGH in periplasmic space when using SufI compared to control.

Optimization of the expression of phaC2 encoding poly (3-hydroxyalkanoate) synthase from Pseudomonas aeruginosa PTCC1310 in Fad B deleted Escherichia coli.

BACKGROUND: Poly3-hydroxyalkanoates (PHAs) are potential candidates for the industrial production of biodegradable plastics. Therefore, in the present study, expression and activity of one of the enzymes involved in the PHA synthesis, phaC2 (isolated from Pseudomonas aeruginosa PTCC1310), were investigated in Fad B deleted Escherichia coli. MATERIALS AND METHODS: The inserts obtained from recombinant pTZ57R plasmids were ligated into the pGEX-5x-1 expression vector and then transformed into Fad B deleted E. coli cells using the heat shock method. This protein was then expressed using isopropyl beta-d-thiogalactoside (IPTG) as an inducer. By changing expression conditions such as IPTG and glucose concentration, time and temperature of incubation with IPTG, the expression conditions were optimized. RESULTS: The optimum condition for the expression of this enzyme was: 1.5 mM IPTG, 1 mM glucose, incubated at 37°C for 2 hours. CONCLUSION: We obtained functional expression of the phaC2 gene and investigated various conditions that could influence the expression of protein to optimize production of PHA synthase enzymes. This would allow us to study PHA production in large quantities.

Isolation and Characterization of a New Thermoalkalophilic Lipase from Soil Bacteria.

Lipases are diversified enzymes in their properties and substrate specificity, which make them attractive tools for various industrial applications. In this study, an alkalinethermostable lipase producing bacteria were isolated from soil of different regions of Isfahan province (Iran) and its lipase was purified by ammonium sulfate precipitation and ion exchange chromatography. To select a thermoalkalophil lipase producing bacterium, Rhodamine B and Horikoshi media were used and the strain that can grow at 45° Cwas selected. The isolated strain was identified using microbial and biochemical tests. One strain showed an orange colored zone on plate and grew on Horikoshi plate. Microbial and biochemical tests showed that the isolated strain was Bacillus subtilis, a Gram positive rod. In PCR, an expected band was obtained with about 371 bp. The activity of the purified lipase was 10.2 folds that of the standard enzyme using ammonium sulfate precipitation and ion exchange chromatography. The molecular weight of lipase determined by SDS-PAGE electrophoresis, was 21 and 35 KDa. Existence of two bands in SDS-PAGE electrophoresis and low amount of obtained purified enzyme highlights the necessity of optimization of purification and concentrating process.

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.

Association of KCNJ11 (E23K) gene polymorphism with susceptibility to type 2 diabetes in Iranian patients.

BACKGROUND: Type 2 diabetes (T2D) is a multifactorial disease with susceptibility of several genes that are related to T2D. Insulin secretion pathway starts with potassium channels in pancreatic beta cells. KCNJ11 gene encodes ATP-sensitive potassium channel subunits. Some studies suggested that KCNJ11 (E23K) mutation increases the risk of T2D. Therefore, present study was designed to investigate the association between E23K polymorphism of KCNJ11 gene and type 2 diabetes mellitus (T2DM) in the Iranian population. MATERIALS AND METHODS: The type of study was case-control and 40 unrelated subjects, including 20 healthy controls and 20 diabetic patients were recruited (diagnosed based on American Diabetes Association criteria). Blood samples were used for isolation of genomic deoxyribonucleic acid (DNA). Having extracted the genomic DNA from human blood leukocytes by means of High Pure PCR Template Preparation Kit, PCR-restriction fragment length polymorphism method was used to detect KCNJ11 E23K gene polymorphism. BanII restriction enzyme was used for digestion. Data were analyzed using Chi-square or Fisher exact test or independent t-test, as appropriate. P < 0.05 was considered. RESULTS: We found that the carrier homozygous for KK genotype are susceptible to T2D (0.049) and in patients the frequency of K allele was higher than control subjects (0.048). CONCLUSION: The present study suggests that KCNJ11 (E23K) gene polymorphism is associated with T2DM.

Inactivation of aprE Gene in Bacillus subtilis 168 by Homologus Recombination.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Polymerase chain reaction amplification of a GC rich region by adding 1,2 propanediol.

BACKGROUND: Apolipoprotein E (ApoE) is one of the most important carriers of lipids in mammalians. The gene for this lipoprotein (ApoE) is located on chromosome 19 which is related with the pathogenesis of some nervous system disease. ApoE gene is identified as a high guanine-cytosine (GC) content fragment. Detection and amplification of these templates are extensively laborious and baffling. The aim of this study was to find a practical and feasible method for the amplification of the number of GC rich genes such as ApoE. MATERIALS AND METHODS: We experimented with simple polymerase chain reaction (PCR), nested PCR and PCR with 1-2 propanediol, dimethylsulfoxide (DMSO), and ethyleneglicol as additive substances to enhance the amplification ApoE gene and used the 40 samples of the human whole blood were collected in test tubes with a pre-treatment of ethylene diaminetetraacetic acid. RESULTS: According to our observations, presence of 1-2 propanediol, DMSO, and ethyleneglicol as additive substances resulted to enhanced amplification of ApoE gene. Addition of 1-2 propanediol showed the best results, caused optimization and revealed more specific and sharp bands. CONCLUSION: According to our findings 1-2 propanediol are the best organic reagent for improving the amplification of ApoE gene. Optimization procedure for each GC rich sequence is recommended to be performed separately in order to identify which of the additive agent is more efficient and applicable for a particular target.

Isolation and characterization of novel thermophilic lipase-secreting bacteria

The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered.

Comparison of the cytoplasmic and periplasmic production of reteplase in Escherichia coli.

Reteplase is the recombinant type of tissue plasminogen activator variant. In this study, preplasmic and cytoplasmic (as inclusion body: IBs) production and activity of recombinant reteplase in E. coli were investigated and compared using a pET system (pET22b and pET15b). The cDNA of reteplase was cloned by polymerase chain reaction (PCR) amplification, sequenced, inserted into the vector pET 22b and pET15b, and expressed using isopropyl ß-D-1-thiogalactopyranoside (IPTG). The recombinant plasmid was expressed in the form of inclusion body in pET 15b and in periplasmic space in pET22b. The obtained results of inclusion body extraction from recombinant pET22b (rpET22b) and recombinant pET15b (rpET15b) plasmids using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a band of ~39 kD. However, the obtained results of periplasmic space extraction from rpET22b plasmid showed a very weak band, while cytoplasmic expression of reteplase (pET15b) produced a strong protein band confirmed with Western blotting. Consequently, our results demonstrated that the cytoplasmic expression system is efficient for the production of reteplase protein in prokaryote systems and a high amount of reteplase was obtained from the expressed proteins in the form of IBs. The obtained activity of rpET15b plasmid showed a higher enzyme absorbance in comparison to rpET22b plasmid. This suggests rpET15b as an appropriate candidate for reteplase production.

Isolation and characterization of novel thermophilic lipase-secreting bacteria.

The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered.

Cloning and Expression of Randomly Mutated Bacillus subtilis α-Amylase Genes in HB101.

The aim of this study was to isolate and express the randomly mutated α-amylase gene from B. subtilis strain 168. BS168F: 5'-gtgtcaagaatgtttgc-3' and BS168R: 3'-gttttgttaaaagatga-5' primers were used to amplify the amylase gene using the following cycle in error-prone PCR method: 94°C for 30 s, 40°C for 2 min, and 72°C for 2 min in 30 cycles that were followed with 72°C for 2 min as a post cycle. E. coli XL1 blue was used as host for plasmid construction. Amylase enzyme activity assay was performed using continuous spectrophotometric procedures. Results of sequencing showed that sequence was cloned from the first ATG and with the correct open reading frame. Having confirmed the integrity of the insert, the gene was ligated into expression vector pET-15b and then further confirmed using digestion analysis. Amylase activity showed 3 clones with higher enzymatic activity compared with the wild type. Error-prone PCR method produced a mutated gene that provides amylase activity much higher than that of wild type. Sequencing the mutated genes should shed light on the important region of the genes that could be manipulated in future studies.

Polymorphism of Apo lipoprotein E gene and the risk of multiple sclerosis.

BACKGROUND: Apolipoprotein E (ApoE) gene encodes an important protein in reforming injuries of central nervous system (CNS). It is assumed that various ApoE alleles may be functionally different. The purpose of this study was to investigate the distribution of ApoE genotypes in multiple sclerosis (MS) patients in a small cohort of Iranians. METHODS: In this case-control study, blood samples of patients and healthy volunteers were collected (n = 40) from Neurology Clinic of Alzahra Medical Complex. The ApoE genotypes were determined using DNA extracted from the samples by polymerase chain reaction (PCR) techniques followed by digestion with HhaI restriction enzyme. The results were adjusted for age of MS onset, sex, expanded disability status scale (EDSS), and type of MS (primary or secondary progressive). Results were statistically analyzed using chi-square test. RESULTS: The ApoE3/E3 genotype was detected in the majority of MS patients and the control group. Frequency distribution of E4 allele did not differ significantly between the two groups. There was no difference between ApoE allele and age of disease onset, sex, expanded disability status, or type of multiple sclerosis. CONCLUSIONS: We found no significant differences in genotype frequency between patients with multiple sclerosis and the control group. Despite the fact that small sample size was a limitation for our study, it seems that ApoE polymorphism may not be useful as a marker for screening patients with multiple sclerosis.

Expression of an innate immune element (mouse hepcidin-1) in baculovirus expression system and the comparison of its function with synthetic human hepcidin-25.

Hepcidin is an innate immune element which decreases the iron absorption from diet and iron releasing from macrophage cell. In contrast to the chemical iron chelators, there has been limited effort applied to the specific use of hepcidin as a new drug for decreasing the iron overload. Hepcidin is produced in different biological systems. For instance, E-coli is used for human hepcidin expression, however, post-translational modification is impaired. We have used a simple baculovirus expression system (BES) to improve the hepcidin folding and activity. Hepcidin Messenger Ribonucleic acid (mRNA) was isolated from mouse liver cells and its complementary Deoxyribonucleic acid (cDNA) was produced and amplified. PFastBac HTB vector was used for recombinant bacmid production. Recombinant baculovirus was produced using SF-9 cell line. The mouse hepcidin-1 protein was expressed in a large quantity and functional tests were performed for this recombinant peptide. The yield of hepcidin in BES was 20 µg/mL and anti-histidine (anti-His) tag antibody was used for the confirmation of hepcidin on western blot nitrocellulose paper. Functional tests showed that mouse hepcidin accumulates iron in the macrophage cell line J774A.1 up to 63%. In addition, our data showed that the mouse hepcidin-1 has less toxicity compared to the synthetic human hepcidin-25 (p = 0.000).

Cloning and expression of a human tissue plasminogen activator variant: K2S in Escherichia coli.

The DNA sequence of Kringle-2 and serine protease domains of the human tissue plasminogen activator (reteplase, K2S) was PCR amplified. This product was then cloned into the expression vector pET15b plasmid. The presence of the insert was confirmed by restriction digestion, PCR and determination of the nucleotide sequence. By using isopropyl beta-D thiogalactopyranoside (IPTG), reteplase was induced in E. coli BL21 cells and analyzed using polyacrylamide gel electrophoresis (PAGE).