Optimization of expression, purification and secretion of functional recombinant human growth hormone in Escherichia coli using modified staphylococcal protein a signal peptide.

BACKGROUND: Human Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the Escherichia coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space. RESULTS: In this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISA-based method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm. CONCLUSIONS: Since the native signal protein peptide of S. aureus protein A was not able to deliver hGH to the extracellular space, it was modified using bioinformatics tools and fused to the n-terminal region of hGh to show that the redesigned signal peptide was functional.

Evaluation of serotonin receptors (5HTR2A and 5HTR3A) mRNA expression changes in tumor of breast cancer patients.

Background: Several studies have proven the pattern of neurotransmitters, especially serotonin, in carcinogenesis and tumor development. Several studies have also shown that changes in serotonin receptors, especially 5HTR2A and 5HTR3A, can play an important role in incidence of cancers. This study was conducted to investigate changes in mRNA expression of 5HTR2A and 5HTR3A receptors in the breast tumor tissue compared to their marginal zone. Methods: In this study, tissue samples were obtained from 40 female patients with breast cancer. Entire RNA was obtained from the tissues and cDNA synthesis was performed. Finally, real ime PCR technique was performed to investigate the gene expression variation of both 5HTR2A and 5HTR3A. To analyze the results of real time PCR, both ΔΔCt and 2-ΔΔCt equations were used. All statistical analyses were performed using the SPSS 18 software and R-Studio 1.0.136. P values less than 0.05 (p<0.05) and 0.001 (p<0.001) were considered statistically significant. Results: The results showed increased expression of 5HTR2A and 5HTR3A genes in tumoral tissues of patients with breast cancer compared to their marginal tissues, where the 5HTR2A and 5HTR3A genes expression in tumor tissue was 3.12 and 3.24 times more than that of the marginal zone, respectively. Conclusion: The results indicated an increase in the mRNA expression of serotonin receptors (5HTR2A and 5HTR3A) in the tumor tissue compared to the marginal zone, which due to the mitogenic nature of these receptors, is likely to induce more proliferation of cancer cells.

The first report on molecular cloning, functional expression, purification, and statistical optimization of Escherichia coli-derived recombinant Ficin from Iranian fig tree (Ficus carica cv.Sabz).

The enzyme ficin, abundantly found in the leaves of the common Fig (Ficus carica. L), is a cysteine protease of the plant endopeptidase family. In terms of activity, this enzyme mimics the activity of the papain enzyme. However, the enzyme is more acidic than papain and binds with higher efficiency to its substrate. Ficin is widely used in the food and pharmaceutical industry along with the medical diagnosis. To date, there are no available data on cloning and recombinant production of various isoforms of ficin. In the present study, after the cloning process and optimized expression of ficin in E. coli BL21, by means of the central composite design (CCD) and approach-based response surface methodology (RSM), the recombinant protein was purified using the Ni-sepharose column and gel filtration. The activity of ficin was determined by its ability to hydrolyze the bovine casein enzyme as a substrate. These results showed the presence of different isoforms of ficin in this cultivar that they are distinct in terms of DNA coding sequences. The optimum conditions for maximum production of the recombinant ficin enzyme in E. coli were as follows; a cell density of 1.25, post-induction time 7 h, 10% (w/v) lactose concentration, and shaking at 115 rpm at 24 °C. The concentration of purified product was reported to be 0.27 mg/ml. The optimization procedures increased the amounts of ficin production by approximately 3 folds (0.67 mg/ml) compared with the expiration level (in the absence of optimization). Also, our findings showed that the recombinant ficin was able to hydrolyze casein, denoting the functionality of the enzyme when used in-vitro. The pitfall of cutting-off the young branches of the common fig tree to purify the enzyme from the young shoots was successfully solved in this study.

A comprehensive review on staphylococcal protein A (SpA): Its production and applications.

The Staphylococcus aureus protein A (SpA) can be obtained through the culture of wild-type S. aureus and also as a recombinant protein in safe bacterial hosts. Several methods have been used to purify SpA among which ion-exchange chromatography, affinity chromatography, gel filtration, and per aqueous liquid chromatography (PALC) are common. SpA has a wide range of biochemical, biotechnological, and medical applications and is most commonly used in test methods such as immunoprecipitation, enzyme-linked immunosorbent assay, and Western blotting. SpA has also been widely utilized in pharmaceutical applications to bind to immune complexes and serum immunoglobulins. SpA also directly binds to the B-cells preventing initiation of infectious diseases as well as having a role in the development of various autoimmune diseases. This review considers different applications of SpA in biotechnology and its novel clinical application for effective treatment of autoimmune diseases. It also discusses various strategies for expression and purification of the SpA including types of column chromatography that are commonly used in protein purification and developing SpA surface display technologies. Finally, this review highlights the potential and novel applications of SpA immobilization, SpA typing, protein engineering for further development of immunological and biochemical research, and also application of SpA as a diagnostic biosensor.

Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma.

PURPOSE: Methylated cell-free fetal DNA (cffDNA) in maternal plasma can potentially be used as a biomarker for accurate noninvasive prenatal testing (NIPT) of fetal disorders. Recovery and purification of cffDNA are key steps for downstream applications. In this study, we aimed to developed and evaluated different aspects of an optimized method and compared its efficiency with common methods used for extraction of methylated cffDNA. METHODS: Single factor experiments, Plackett-Burman (PB) design, and response surface methodology (RSM) were conducted for conventional Triton/Heat/Phenol (cTHP) method optimization. The total cell-free DNA (cfDNA) was extracted from pooled maternal plasma using the optimized method called the Triton/Heat/Phenol/Glycogen (THPG), cTHP method, a column-based kit, and a magnetic bead-based kit. In the next step, methylated cfDNA from the extracted total cfDNA was enriched using a methylated DNA immunoprecipitation (MeDIP) kit. Real-time quantitative polymerase chain reaction was performed on the RASSF1 gene and hyper region to determine the genomic equivalents per milliliter (GEq/ml) values of the methylated cfDNA and cffDNA, respectively. RESULTS: The optimum values of the significant factors affecting cfDNA extraction from 200 µl of plasma were 3% SDS, 1% Triton X-100, 0.9 µg/µl glycogen, and 0.3 M sodium acetate. The GEq/ml values of methylated cffDNA extracted using the THPG method were significantly higher than for the tested extraction methods (p < 0.001). CONCLUSIONS: Our results indicate that the THPG method is more efficient than the other tested methods for extraction of low copy number methylated cffDNA from a small volume of maternal plasma.

Covalent Immobilization of Protein A on Chitosan and Aldehyde Double-Branched Chitosan as Biocompatible Carriers for Immunoglobulin G (Igg) Purification.

High-affinity ligands, such as protein A, can be used to develop biocompatible matrices for antibody purification. In this paper, two methods were used for immobilization of protein A on the chitosan. In the first approach, amino groups of chitosan beads were functionalized with tris(2-aminoethyl)amine to produce amine double-branched moieties, which were subsequently activated with glutaraldehyde. In the second approach, chitosan beads were directly modified by glutaraldehyde to produce aldehyde groups. Structural characterization and successful modification of the functional groups on the supports were confirmed by scanning electron microscopy, FTIR spectroscopy and elemental analysis. Covalent immobilization of protein A was then performed on the surface of both supports. The immobilization yield was determined by using fluorescence spectroscopy, showing almost 15% increased capacity for the double-branched derivatized chitosan. The Immunoglobulin G (IgG) purification ability of the double-branched support was also almost 1.7-fold higher than the monoaldehyde derivative at the same condition.

Designing a novel signal sequence for efficient secretion of Candida antarctica lipase B in E. coli: The molecular dynamic simulation, codon optimization and statistical analysis approach.

Lipases represent an important industrial biocatalysts group displaying enantioselectivity, high stability in solvents and substrate specificity. A commonly used commercial enzyme for synthesis of organic materials is Candida antarctica lipase B (CALB). Its Industrial production involves cost-effective purification of large amounts of microbially-produced macromolecules. Hence, great focus is now placed on periplasmic secretion and storage. Accordingly, we designed and constructed a suitable signal peptide for secretion of lipase using a newly developed software. Molecular dynamic simulation was performed to compare structural states of native signal peptide of Staphylococcus aureus protein A (nSpA) and its modified counterpart (mSpA). Furthermore, the effect of these two peptides in binding to the signal peptidase I (SPase I) was studied. Simulation data confirmed experimental results showing that secondary structure of the mSpA binding region and the binding site of the SPase I create a more stable interaction relative to native SpA. Subsequently, response surface methodology (RSM) was employed to improve secretion. Lactose concentration, induction time and temperature were identified as essential parameters to optimize the expression and periplasmic secretion of CALB. mSpA increased CALB expression levels by 2.1-fold relative to the control, which further confirmed efficient secretion of the mature enzyme through the Sec-dependent pathway.

Molecular Engineering of the Geobacillus stearothermophilus α-Amylase and Cel5E from Chlostridium thermocellim; In Silico Approach.

BACKGROUND: Considering natural thermal stability, Geobacillus stearothermophilus amylase and Cel5E from Clostridium thermocellum are good candidates for industrial applications. To be compatible with the industrial applications, this enzyme should be stable in the high temperatures, so any improvement in their thermal stability is valuable. OBJECTIVES: Using in silico approach and identifying point mutations in the structure amylase of G. stearothermophilus and Cel5E from C. termocellum we tried to increase thermal stability of the enzymes along with their catalytic activity to reach a new industrial amylase with higher thermostability and an improved function. MATERIALS AND METHODS: In this study we predicted the 3D structure of the enzymes, then simulated the molecular docking study using MolDock, PLANTS, and Lamarkian genetic algorithm as scoring functions for the docking and in silico engineering of the protein aiming to increase the thermal stability and catalytic activity. RESULTS: A series of thermal stability increasing point mutations were exerted around the active site of the enzyme, then by docking procedure, the binding affinity was measured and finally a list of mutations which theoretically improved the increased thermal stability as well as catalytic activity were proposed. CONCLUSIONS: Based on the in silico results obtained the modified enzymes seems to be suitable candidates for considering in both laboratory and industrial scales.

Molecular docking based screening of Listeriolysin-O for improved inhibitors.

Listeriolysine-O (LLO) is a 50KDa protein responsible for Listeria monocytogenes pathogenicity. The structure of LLO (PDB ID: 4CDB) with domains D1 to D4 is known. Therefore, it is of interest to identify conserved regions among LLO variants for destabilizing oligomerization (50 mer complex) of its monomers using appropriate inhibitors. Therefore, it is of interest to identify suitable inhibitors for inhibiting LLO. Previous reports suggest the use of flavanoids like compounds for inhibiting LLO. Our interest is to identify improved compounds to destabilize LLO oligomerization. We used a library (Zinc database) containing 200,000 drug-like compounds against LLO using molecular docking based screening. This resulted in five hits that were further analyzed for pharmacological properties. The hit #1 (2-methyloctadecane- 1, 3, 4-triol) was further refined using appropriate modifications for creating a suitable pharmacophore model LLO inhibition. The modified compound (1-(4-Cyclopent-3-enyl-6, 7-dihydroxy-8-hydroxymethyl-nona-2, 8-dienylideneamino)-penta-1,4-dien-3-one) shows fitting binding properties with LLO with no undesirable pharmacological properties such as toxicity.

Virtual screening following rational drug design based approach for introducing new anti amyloid beta aggregation agent.

Amyloid ß (Aß) sheets aggregations is the main reason of Alzheimer disease. The interacting areas between monomers are residue number 38 to 42. Inhibition of interaction between Aß molecules prevents plaque formation. In the present study, we have performed a high-throughput virtual screening among ZINC database and top 1000 hits were checked again regarding binding affinity by AutoDock software. Top 4 successive second step screening hits was considered for drug design purpose against aggregation site of Aß molecules. The toxicity and pharmacological properties of new designed ligands was assessed by PROTOX and FAFdrugs3 webservers. Several steps of modifications performed in the structures of hit#1 and hit#2 and finally new designed ligand based on hit 1, 1-RD-3 (3-[(Z)-6-Hydroxy-4-{[5-(2-methoxyethyl)-6-methyltetrahydro-2H-pyran-2-yl]methyl}-1-methyl-3-hexenyloxy]tetrahydro-2Hpyran- 4-ol) and a designed ligand based on hit 2, 2-RD-2 (6-(Hydroxymethyl)-4-{5-hydroxy-6-methyl-4-[(3- methylcyclohexyl)methyl]tetrahydro-2H-pyran-2-yloxy}tetrahydro-2H-pyran-2,3,5-triol) could successfully pass pharmacological filters. The LD50 of 37000 mg/kg for 1-RD-3 and 2000 mg/kg for 2-RD-2 indicates that the designed ligands can be considered as new candidates for anti Aß aggregation to treat Alzheimer's disease. Interestingly, after performing several modification steps still a considerable binding affinity of -9.3 kcal/mol for 1-RD-3 and -9.8 kcal/mol for 2-RD-2 still remained. Theoretically, the new designed molecules can reduce the deposition of Aß in the cerebral cortex and as the results the Alzheimer symptoms could be decreased.

Expression and Secretion of Cyan Fluorescent Protein (CFP) in B. subtilis using the Chitinase Promoter from Bacillus pumilus SG2

Background: Improved cyan fluorescent protein (ICFP) is a monochromic, green fluorescent protein (GFP) derivative produced by Aequorea macrodactyla in a process similar to GFP. This protein has strong absorption spectra at wavelengths 426-446 nm. ICFP can be used in cell, organelle or intracellular protein labeling, investigating the protein-protein interactions as well as assessing the promoter activities. Methods: In our previous study, the promoters of two chitinases (ChiS and ChiL) from Bacillus pumilus SG2 were assessed in B. subtilis and their regulatory elements were characterized. In the present study, icfp was cloned downstream of several truncated promoters obtained in the former study, and ICFP expression was evaluated in B. subtilis. Results: Extracellular expression and secretion of ICFP were analyzed under the control of different truncated versions of ChiSL promoters grown on different media. Results from SDS-PAGE and fluorimetric analyses showed that there were different expression rates of CFP; however, the UPChi-ICFP3 construct exhibited a higher level of expression and secretion in the culture medium. Conclusion: Our presented results revealed that inserting this truncated form of Chi promoter upstream of the ICFP, as a reporter gene, in B. subtilis led to an approximately ten fold increase in ICFP expression.

Association of the gene expression variation of tumor necrosis factor-α and expressions changes of dopamine receptor genes in progression of diabetic severe foot ulcers.

OBJECTIVES: Regulation of pro-inflammatory factors such as TNF-α which are secreted by the immune cells through induction of their several receptors including dopamine receptors (especially DRD2 and DRD3) is one of the noticeable problems in diabetic severe foot ulcer healing. This study was conducted to evaluate the alteration of TNF-αin plasma as well as DRD2 and DRD3 changes in PBMCs of diabetics with severe foot ulcers. MATERIALS AND METHODS: Peripheral blood samples were collected from 31 subjects with ulcers, 29 without ulcers, and 25 healthy individuals. Total mRNA was extracted from PBMCs for the study of DRD2, DRD3, and TNF-α gene expression variations. Expression patterns of these genes were evaluated by real-time PCR. Consequently, concentration of TNF-α was investigated in plasma. RESULTS: Significant decrease in gene expression and plasma concentration of TNF-α in PBMCs was observed in both patient groups at P<0.05. These diminutions are correlated to the decrease in the expression of both DRD2 and DRD3 in PBMCs of both patient groups. Also, the same relationship is present between expressions of two new DRD3 transcripts with TNF-α downturn. CONCLUSION: We concluded that DRD2 and DRD3 expression alteration and presence of new DRD3 transcripts can be effective in reduction of TNF-α expression as a pro-inflammatory factor. Performing complementary studies, may explain that variations in DRD2 and DRD3 are prognostic and effective markers attributed to the development of diabetes severe foot ulcers.

Molecular docking based screening of predicted potential inhibitors for VP40 from Ebola virus.

Ebola virus is a member of Filoviridae and cause severe human disease with 90 percent mortality. The life cycle of Ebola contains an assembly stage which is mediated by VP40 proteins. VP40 subunits oligomerize and form ring-structures which are either octamers or hexamers. Prevention of VP40 matrix protein assembly prevents virus particle formation as well as virus budding. In the present study we simulated the biological condition for a single VP40 subunit. Then a library containing 120.000 drugs like chemicals was used as the virtual screening database. Top 10 successive hits were then analyzed regarding absorption, distribution, metabolism, and excretion properties. Moreover probable accessorial human protein targets and toxicity properties of successive hits were analyzed by in silico tools. We found 4 chemicals that could bind VP40 subunits in a manner that by making an interfering steric condense prevents matrix protein oligomerization. The pharmacokinetic and toxicity studies also validated the potential of 4 finlay successive hits to be considered as a new anti-Ebola drugs.

Immobilization of Bioactive Protein A from Staphylococcus aureus (SpA) on the Surface of Bacillus subtilis Spores.

Protein A from Staphylococcus aureus (SpA) is a 40-60 kDa cell-wall component, composed of five homologous immunoglobulin (Ig)-binding domains folded into a three-helix bundle. Each of these five domains is able to bind Igs from many different mammalian species. Recombinant SpA is widely used as a component of diagnostic kits for the detection and purification of IgGs from serum or other biological fluids. In this study, purified SpA was adsorbed and covalently linked to Bacillus subtilis spores. Spores are extremely stable cell forms and are considered as an attractive platform to display heterologous proteins. A sample containing about 36 µg of SpA was covalently immobilized on the surface of 4 × 10(10) spores. Spore-bound SpA retained its IgG-binding activity, even after seven consecutive binding and washing steps, suggesting that it can be recycled and utilized several times. FACS analysis revealed that spores with covalently attached SpA had significantly improved fluorescence intensities when compared to those of spores with adsorbed SpA, suggesting that the covalent approach is more efficient than sole adsorption regarding protein attachment to the spore surface.

Molecular docking based virtual screening of compounds for inhibiting sortase A in L.monocytogenes.

Listeriosis is considered as an important public health issue. Sortase A (srtA) is an enzyme with catalytic role in L. monocytogenes that breaks the junction between threonine and glycine in the LPXTG motif (a key motif in internalin A (InlA) that plays an important role in listeriosis). Inactivation of srtA was shown to inhibit anchoring of the invasion protein InIA. This is in addition to inhibiting peptidoglycan-associated LPXTG proteins. Therefore, it is of interest to inhibit strA using potential molecules. Here, we describe the design of an inhibitor with high binding affinity to srtA with the ability to prevent the attachment of srtA to the LPXTG proteins such as InIJ. A homology model of Listeria monocytogenes Sortase A was developed using MODELLER (version 9.12). We screened StrA to 100,000 drug-like ligands from the Zinc database using Molecular docking and virtual screening tool PyRX). Pharmacokinetic analysis using the FAFDrugs(3) web server along with ADME and toxicity analysis based on Lipinski rule of five were adopted for the screening exercise followed by oral toxicity check using PROTOX (a server) for every 10 successive hits. The results from PROTOX server indicated that Lig #1 (with LD50 of 2000mg/kg) and Lig #7 (with LD50 of 2000mg/kg) have toxicity class 4 and Lig #3 (with LD50 of 14430mg/kg) has toxicity class 6. Subsequent modifications of these structures followed by FAFDrugs(3) analysis for high bioavailability value selected Lig #7 according to Lipinski rules of five. Thus, Lig #7 with IUPAC name ((R)-4-{(S)-1-[(S)-2-Amino-4-methylvaleryl]-2-pyrrolidinyl}-1-[(S)-1-(ethylamino) carbonyl-propylamino] -2-propyl-1, 4- butanedione) is suggested as a potential candidate for srtA inhibition for further consideration.

An Alternative Bacterial Expression System Using Bacillus pumilus SG2 Chitinase Promoter.

BACKGROUND: Chitin is an abundant natural polysaccharide found in fungi, algae, and exoskeleton of insects. Several bacterial species are capable of utilizing chitin as their carbon source. These bacteria produce chitinases for degradation of chitin into N-acetyl-D-glucosamine. So far, regulation of the chitinase encoding genes has been studied in different bacterial species. Among Bacillus species, B. pumilus strain SG2 encodes two chitinases, ChiS and ChiL. The promoter region of chiSL genes (P chiS ) is mainly regulated by the general carbon catabolite repression (CCR) system in B. subtilis due to the presence of a catabolite responsive element (cre). OBJECTIVES: Use of P chiS in constructing an inducible expression system in B. subtilis was investigated. MATERIALS AND METHODS: In the first step, complete and shortened versions of P chiS were inserted upstream of the lacZ on a pBS72/pUC18 shuttle plasmid. The ß-galactosidase activity of B. subtilis carrying one of the relevant plasmids was measured in the presence of different carbon sources. RESULTS: An expression system based on the chitinase promoter of B. pumilus SG2 was established. Modification of P chiS and the culture medium resulted in production of ß-galactosidase in B. subtilis up to 1,800 Miller unit (MU) activity. CONCLUSIONS: The chitinase promoter developed in this study, has potential to be used in an expression vector that could be induced by chitin. In addition, compared to the other inducers like IPTG and lactose, chitin is definitely cheaper and more available as an inducer.

Optimization of extracellular truncated staphylococcal protein A expression in Escherichia coli BL21 (DE3).

Staphylococcal protein A (SpA) plays an important role in Staphylococcus aureus pathogenesis. The recombinant SpA is also widely used in biotechnology to purify polyclonal and monoclonal immunoglobulin G antibodies. In this study, expression and secretion of a truncated form of SpA containing five immunoglobulin-binding domains using its own native signal sequence were optimized in Escherichia coli. Optimization was carried out using response surface method (RSM), making use of the interaction between five variables. The initial results revealed that the signal peptide from S. aureus was recognized in E. coli and the resulting SpA was expressed and secreted into the medium. Compounds, such as glycine, affected the secretion of SpA into the culture medium. The central composite design experiment showed that the optimum conditions for the maximum expression of recombinant truncated SpA in E. coli included 10% (w/v) lactose, 1.77% (w/v) glycine, induction time of 11 H, an optical density (600) of 1.1, and a temperature of 33 °C. Optimization using RSM resulted in a fivefold increase in the secretion of SpA. To date, this is the first study of its kind regarding the definite influence of glycine concentration and duration of the cultivation period on the secretion of SpA.

Improving the chitinolytic activity of Bacillus pumilus SG2 by random mutagenesis.

Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and ChiL that were induced by chitin and secreted into the supernatant. The present work aimed to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on chitin agar and subsequent formation of halos, the mutated strains were examined for degradation of chitin under different conditions. A mutant designated AV2-9 was selected owing to its higher chitinase activity. To search for possible mutations in the whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced. Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9 strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL). The results demonstrated that a single base change had occurred in the ChiL sequence in AV2- 9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed, and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30% higher catalytic activity under all the conditions tested. The results obtained in this study showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics analysis was performed to predict changes in the stability of proteins caused by mutation.

Differential proteome analysis of a selected bacterial strain isolated from a high background radiation area in response to radium stress.

The present study describes the response of a bacterial strain, isolated from a hot spring in an area with the highest levels of natural radiation, under radium ((226)Ra) stress. The bacterium has been characterized as a novel and efficient radium biosorbent and identified as a variant of Serratia marcescens by biochemical tests and molecular recognition. In order to gain insights into key cellular events that allow this strain to survive and undergo (226)Ra adaptation and biosorption, the strain was tested under two experimental conditions of 1000 and 6000 Bq (226)Ra stress. A proteomic approach involving two-dimensional polyacrylamide gel electrophoresis and mass spectrometry was used to identify the differentially expressed proteins under (226)Ra stress. Functional assessment of identified proteins with significantly altered expression levels revealed several mechanisms thought to be involved in (226)Ra adaptation and conferring resistant phenotype to the isolate, including general stress adaptation, anti-oxidative stress, protein and nucleic acid synthesis, energy metabolism, efflux and transport proteins. It suggests that this strain through evolution is particularly well adapted to the high background radiation environment and could represent an alternative source to remove (226)Ra from such areas as well as industrial radionuclide polluted wastewaters.