Dodecyl Sulfate-polyacrylamide Gel Research Articles

Relative Protein Profile of Philippine Cinnamomum (Schaeffer) Using Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Philippine Cinnamomum are trees with aromatic leaves and bark. It has several economic and medicinal uses, but concerns about protein profile are limited. Thus, this study was conducted to determine the relative protein component of the species by Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE). A total of 27 cinnamon species were run in Sodium Dodecyl Sulphate - Polyacrylamide Gel Electrophoresis (SDS-Page) with LSB (Laemli Sample Buffer) and Tris HCI (1:1) as sample buffer. Determining protein profiles is a tool for the systematic classification of the species. Results showed that Philippine Cinnamomum leaf samples have the same component proteins. The SDS-PAGE provided a similar protein banding pattern, although the same bands' intensity varies. Pronounced protein bands were observed corresponding to 50 to 75 kDa and 25 kDa, with additional minor bands indicating a higher protein content. The results in SDS-PAGE showed that the component proteins of Cinnamomum leaves were comparable regardless of geographical source and different stages of growth of the samples. However, the concentration of some component proteins may vary, as indicated by the difference in intensity of protein bands. Sodium dodecyl sulphate Polyacrylamide gel electrophoresis (SDS-PAGE) is a tool in the biochemical method of determining the relative protein profile of Cinnamomum species. It can be used to classify plants up to the species level. Based on the study results, Philippine Cinnamomum has a higher protein content that can be used in culinary, medicinal, and pharmaceuticals.

Plant proteases and anti-bacterial substances in Allium sativum L. varieties

Allium sativum L. protease still remains largely understudied although new varieties of garlic appear quite often, e.g., lanang garlic. This study tested the antibacterial effect of garlic and the effectiveness of various A. sativum proteases as meat tenderizers. 
 The research involved powder extracts of four varieties of A. sativum: kating, lanang, black garlic, and sin-chung. The degradation kinetics was defined based on the Lineweaver-Burk equation. The degradation zones were measured using sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE). Scan electron microscopy served to test the changes in meat connective tissue.
 Lanang demonstrated the largest inhibition zones against Escherichia coli (9.75 ± 0.15 mm) and Staphylococcus aureus (1.04 mm). Sin-chung protease degraded beef protein with the highest Vmax of 0.1818 μg/μL/min at 10–22 KDa (small peptide, troponin C, and troponin I), 25–40 KDa (myosin light chain, troponin T, α and β tropomyosin, actin), and 100–140 KDa (protein C). The same garlic variety degraded mutton meat protein at 10–17 KDa (small peptide) and 25–40 KDa (myosin light chain, troponin T, α and β tropomyosin, actin) with Vmax of 0.1135 μg/μL/min.
 All four A. sativum proteases proved to be quite effective meat tenderizers.

Plasmid and Total Protein Analyses of Extended Spectrum Beta Lactamase Producing Bacteria from Periplaneta americana (Coackroaches)

Background: The importance of plasmid-encoded Extended Spectrum Beta Lactamase (ESBL) organisms in the dissemination of multi-drug resistance and complicated infections are contributory factors to treatment failure. Despite these, there is a paucity of information regarding plasmid-borne associated infection from primary sources including cockroaches while studies correlating total protein and plasmid-encoded ESBL are also limited especially in cockroaches. The objective of this study was therefore aimed at determining the presence of plasmid in the ESBL organisms from cockroaches as well as their total protein profiles.
 Materials and Methods: Bacterial isolation, characterization and molecular identification were carried out in our previous work following standard recommended techniques. These molecularly identified organisms which were delineated into twenty-two (22) were further subjected to plasmid and total protein analyses using alkaline lysis and Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) respectively.
 Results: Results obtained identifies 18(81.8%) of the ESBL isolates to be harbouring plasmids of different molecular weights (2-21.5kbp). The total protein profile of the plasmid-encoded ESBL and the non-plasmid encoded ESBL reveals differential protein expression patterns except for two non- plasmid encoded ESBL isolates that have similar patterns as that of plasmid-encoded ESBL organisms (isolates 2 and 3).
 Conclusion: There is a need to monitor the primary sources of infection in the epidemiological distribution of resistance. Improving environmental sanitation through proper disinfection of carrier insects may be a means to curtailing such spread.

OPTIMIZATION OF ASPERGILLUS TUBINGENSIS GROWTH FOR CELLULASE PRODUCTION

Celluloses are the basic component of plant cell walls which are difficult to break however, cellulase enzymes have the ability to degrade the cellulose into glucose. The use of celullase enzymes is spreading widely particularly in the paper, pulp and food industries to reduce biowaste. The purpose of this study was to optimize the production of cellulase enzymes by using fungal strain Aspergillus tubingensis. The fungal strain Aspergillus tubingensis was extracted from putrid fruits and vegetable samples collected from the local fruit market of Pattoki in the District Kasur. The identification of fungus was carried out using microscope. Corn Stover was used as a substrate for cellulase production. Different parameters were used for the optimization of fungus growth such as substrate level, substrate particle size, temperature, pH, moisture, urea and glucose. Cellulase enzymes were purified by salting out and gel filtration techniques. The optimum substrate level, substrate particle size, temperature, pH, glucose, urea and moisture were analyzed as 5g, 40mm, 40°C, 4.5pH, 0.5g, 0.04g and 60% respectively. The maximum activity reveled by cellulase enzyme at different optimized parameters were reported in U/mL/min. The cellulase molecular weight was determined 71 kDa by Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis. It was concluded that the study will help to a way for the scientists for the maximum production of cellulase from Aspergillus tubingensis using Corn stover as substrate and most cost-effective Solid State Fermentation method.

Tailoring Butyl Methacrylate/Methacrylic Acid Copolymers for the Solubilization of Membrane Proteins: The Influence of Composition and Molecular Weight.

Low-molecular weight (MW) amphiphilic copolymers have been recently introduced as a powerful tool for the detergent-free isolation of cell membrane proteins. Herein, a screening approach is used to identify a new copolymer type for this application. Via a two-step ATRP/acidolysis procedure, a 3 × 3 matrix of well-defined poly[(butyl methacrylate)-co-(methacrylic acid)] copolymers (denoted BMAA) differing in their MW and ratio of hydrophobic (BMA) and hydrophilic (MAA) units is prepared. Subsequently, using the biologically relevant model (T-cell line Jurkat), two compositions of BMAA copolymers are identified that solubilize cell membranes to an extent comparable to the industry standard, styrene-maleic acid copolymer (SMA), while avoiding the potentially problematic phenyl groups. Surprisingly, while only the lowest-MW variant of the BMA/MAA 2:1 composition is effective, all the copolymers of the BMA/MAA 1:1 composition are found to solubilize the model membranes, including the high-MW variant (MW of 14000). Importantly, the density gradient ultracentrifugation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting experiments reveal that the BMA/MAA 1:1 copolymers disintegrate the Jurkat membranes differently than SMA, as demonstrated by the different distribution patterns of two tested membrane protein markers. This makes the BMAA copolymers a useful tool for studies on membrane microdomains differing in their composition and resistance to membrane-disintegrating polymers.

ASSESSMENT OF NUTRITIONAL RELEVANCE OF DIPLAZIUM ESCULENTUM BY QUALITATIVE PHYTOCHEMICAL AND OVERALL PROTEIN PROFILING

Diplazium esculentum is one of the very well-liked edible ferns, a common pteridophyte. Very few reports are published about the plant from India. Pteridophytes are acquainted for their biological and medicinal properties for long time but very fewevidences are seen about wetland pteridophytes. The present work focuses on assessment of nutritional relevance of D. esculentum collected from Alappuzha District of Kerala in India, by qualitative phytochemical and overall protein profiling. Chloroform and methanolic extracts of leaves of D. esculentum are used for both assays. The results showed the presence of vital phytoconstituents on qualitative analyses that included Alkaloids, Saponins, Flavonoids, Steroids, Phenols, Cardiac glycosides and Carbohydrates. On protein profiling by Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS PAGE), 5 distinct bands corresponding to molecular weights ~250 kDa, ~130 kDa, ~100 KDa, ~55 KDa and ~15 KDa which showed nutritional importance of the fern, Diplazium esculentum. The future perspectives of study may include the quantitative estimation of phytoconstituents and proteins that paves the way for the incredible health benefits of the fern for humans.

The Assembly of Bacteriophage Functional Enzymatic Models in Association with E. coli Proteins’ Profiles

Introduction: The invasion of bacteriophage on the associated host bacterium depends on their receptors’ orientation that adsorb them to cell surface. During phage replication a valuable number of proteins acts as lytic enzymes for host puncher at the beginning of the infection and other for burst after lytic cycle compilation. Accordingly, the proteomic relationship among phage and bacterium proteins could easily be studied by their protein profiles analysis. Objective: To detect bacteriophages functional enzymes during lytic cycle. Methods: The isolation and identification of Escherichia coli and their parasitic T7 phage group was done using bacterial culture and common plaque assay techniques. The investigations and protein-protein interactions’ assays were inveterate by proteins profile of phage and bacterium using Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) to find out their molecular weights, where the scaled location of each mobile band was compared to the standards of identified proteins weights in the molecular ladder. Thereafter, Protein model’s assembly and bands migration was done by computer analytical software. Results: Mobilization of the phage’ proteins inside the Two Dimensions (2D) gel ranged between 60 and 12 kDa where a model of 4 main bands with molecular weights of (46, 35, 24 and 14 kDa) is corresponded to the host ones, where pure 9 bands with molecular weight ranged between 96-24 kDa. The computational model analysis showed common shared molecular masses of 47, 34 and 16 kDa on plot area of the phage and the bacterium. Model interpretation confirmed that proteins ranged from 47.7 to 34.3 kDa resembles 43.3% of whole phage’s proteins that assembled the capsid head and the coil, while the molecular weight mass of 22.5 formed the tail’s proteins. The lytic enzymes’ molecular weight was ranged between 18-14 kDa according to the function of the enzyme. The study revealed that the 34 kDa band has the common shared peak between T7 phage group and associated Escherichia coli host. Conclusion: Functional models of analysed proteins during phage assembly, ensures lytic enzymes are built in the capsid head and the lysozyme in the tail, they facilitate the enzymatic decay for bacterial host. This enzymatic function is related to the lytic cycle of the bacteriophages and their phenomenon in employing the bacterial DNA in proteins manufacturing during their replication inside host.

Genetic diversity in the natural population of threatened fruit- bearing plant species Monotheca buxifolia (Falc.) A.D.

Monotheca buxifolia is an ethnomedicinally and economically important threatened fruit bearing plant species in Malakand Division Pakistan. The genetic diversity among the 92 various genotypes of Monotheca buxifolia was carried out using sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE) method. A considerable amount of inter districts genetic diversity (66.70%) was observed among the genotypes of M. buxifolia. Protein profiling was conducted on 12% gel electrophoresis. A total of 6 protein bands were observed in M. buxifolia genotypes. SDS-PAGE practice is a convenient scheme for the examination of both genetic diversity and relationship. Particularly, L-4 and L-5 were monomorphic in the inter districts Monotheca buxifolia genotypes and was recognized as species specific. The remaining other loci were polymorphic. In this investigation, the high inter and intra- districts specific diversity was observed demonstrating SDS-PAGE is an authoritative procedure for categorizing the genetically diverse germplasms in M. buxifolia. The findings from this study could be useful in the identification and selection of suitable M. buxifolia genotypes for future conservation programmes. Today, there is still a need to examine the genetic diversity and protect genetic resources, in particular wild species, for possible benefits in plant conservation programmes. To the best of our knowledge, this is the first ever report that addresses genetic variability in M. buxifolia.

Genetic diversity in threatened plant species Alnus nitida (Spach.) Endel

Alnus nitida (Spach) Endl. is an ethnobotanically important threatened plant species. The genetic diversity among the 50 different genotypes of Alnus nitida was carried out using sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE) characterization. A considerable amount of genetic diversity (90%) was observed among the genotypes of A. nitida. The protein characterization was carried out on 12% gel electrophoresis. A total of 10 protein bands were detected in A. nitida genotypes. SDS-PAGE procedure is a useful method for the investigation of both genetic diversity and phylogenetic relationship. Especially, B-5 was monomorphic in A. nitida genotypes and was considered as species specific. All other bands/loci were polymorphic. These polymorphic bands displayed 12, 16, 72, 88, 2, 44, 84, 54 and 12 percent variation respectively. In the present examination, the high intra-specific diversity was observed representing SDS-PAGE is a powerful tool for determining the genetically diverse germplasms in A. nitida. The results obtained by this study could be helpful in the identification and selection of desired genotypes of Alnus nitida for conservation programmes in future. Today, there is still a need to assess genetic variation and protect genetic resources, especially of wild species for prospective benefits in plant conservation programmes.

Silver Nanoparticle Trypsin Corona Formation and the Impacts on Enzymatic Potency

Herein, the protein corona formation on the spherical metal nanoparticles was studied to investigate the possible effects of silver nanoparticles (AgNPs) on the protein activity and conformation. The digestion capability of trypsin was monitored on the human serum albumin (HSA) at standard enzymatic hydrolysis conditions in the absence and presence of different concentrations of AgNPs. So the ratio of enzyme:HSA, the duration and the temperature of nanoparticle treatment were evaluated. The activity of treated trypsin molecules, in the form of hard (HC) and soft corona (SC) were studied using sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE), and nano liquid chromatography electrospray (ion trap) mass spectrometry (nano LC-ESI/MS, LC-ESI/MS). In addition the characteristics of silver nanoparticles and the formation of HSA corona on the nanoparticle surface were monitored using ultra-violet/visible spectroscopy (UV-Vis), Dynamic light scattering (DLS) and fluorescence spectrophotometry. The results demonstrated that not only the corona formation but also the AgNPs/Trypsin interaction, decreases the hydrolysis potency of trypsin. Furthermore, the encountering of the AgNPs/HSA could influence both nanoparticles and HSA molecule features. Accompanied with fluorescence study, that the HSA secondary structure. Also LC-ESI/MS data revealed the most affected HSA triptics have α-helix structure.

Identification of Candidate Biomarkers for HCV Leading to Hepatocellular Carcinoma Differential Stages From Serum Samples

Background: Hepatocellular carcinoma (HCC) is one of the most deadly liver malignancy found and Hepatitis C virus (HCV) is a prominent risk factor for this disease. Prognosis of HCC is poor; initiate the need of markers to discover therapeutic targets in HCC. Introduction: Clinical staging systems of HCC composed of tumor characteristics along with liver function test are important in prognosis but they are not precise. Molecular profiling can lead to a better understanding of the physiopathology of HCC and can help in the development of novel therapeutic approaches. Methods: 64 HCC serum samples (shifted from HCV) were graded into stage I- IV; along with +ive (3 Hepatitis C) and -ive control (2 healthy persons). Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and differential mRNA expression from serum samples of different HCC stages was confirmed by Real Time Polymerase Chain Reaction (qPCR). Results: HCC serum proteins displayed differential expression of glutathione s-transferase (GST), glypican-3 (GPC3), vitronectin (VTN), and clusterin (CLU) by SDS-PAGE. GST was expressed in -ive control, while GPC3 was found in both -ive and +ive control. The qPCR analysis, display more than 0.07 fold decrease in GST in I-IV HCC stages. The highest increase in HCC stages was observed by GPC3; about 4 fold increase in I-IV stages. VTN show 1.7-3.4 fold; while CLU show 2-3.5 fold increase in four stages of HCC. Conclusion: GPC3, VTN and CLU in combination can be good potential markers for differentiating stages (I-IV) of HCC.

Variation of Storage Proteins in Crimean Populations of Dasypyrum villosum

The objective of the investigation was to study variation of storage proteins in two Crimean populations of Dasypyrum villosum from Beregove of the Bakhchisarai region and from the Tauric Chersonese National Reserve (Sevastopol). Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of seed storage proteins was performed to analyze diversity of high-molecular-weight glutenin subunits encoded by the Glu-V1 locus, as well as ??-gliadin variants on SDS-electrophoregrams encoded by Gli-V1. In the two Crimean populations of D. villosum, eight alleles at the Glu-V1 locus and four Gli-V1 alleles encoding ??-gliadin were identified. The Crimean populations of D. villosum differed significantly in the frequencies of the alleles a, b, and c at the Glu-V1 locus and both had a high frequency of the null allele (k). The populations showed significant differences in frequencies of ??-gliadin variants on SDS-electrophoregrams encoded by Gli-V1, and gene diversity with respect to this marker was higher in the population of Beregove than that in the Chersonese population. ??-Gliadins on SDS-electrophoregrams are a convenient marker system for analysis of D. villosum populations, which can be employed simultaneously with analysis of high-molecular-weight glutenin subunits.

Quantification molecular weight of human beta defensin-2 and human beta defensin-3 from saliva of caries patient

BackgroundDental caries is the most common multifactorial disease in the world. In saliva of caries patients, occur an increase in innate immunity, such as human beta defensin-2 (HBD-3) and human beta defensin-2 (HBD-3). Research on quantification the molecular weight of HBD-2 and HBD-3 in saliva of caries patients has not done yet.ObjectiveDetermine the molecular weight from HBD-2 and HBD-3 from saliva of caries patients using the Western Blot method.MethodThis study was approved by the Health Research Ethical Clearance Commission of Faculty of Dental Medicine Airlangga University No.156/HRECC.FODM/IV/2019. After every individual agreed, saliva sample from 16 children with caries and caries-free in the Dental Hospital Airlangga University was collected in June to July 2019. Male/female aged 9-12 years and DMF-t > 5, taken 5 ml of saliva between 8-10 O'clock, passive-drool method without stimulation. Salivary samples were centrifuged for 20 minutes, 4ºC, with 10.000 rpm. Total protein was calculated using bicinchoninic acid (BCA) Assays before loading into Sodium Dodecyl Sulfate Poly Acrilamide Gel Electrophoresis (SDS-PAGE) to evaluate protein separation based on molecular weight. Furthermore, Western Blot is performed to ensure the protein that appears is HBD-2 and HBD-3 specific protein using primary and secondary antibodies.ResultsTHE molecular weight of HBD-2 and HBD-3 in the saliva of caries patients is 43 kDa, while the salivary free-caries band in the gel is unreadable, thus the molecular weight cannot be determined.The molecular weight of HBD-2 and HBD-3 in the saliva of caries patients calculated using the Western Blot method was 43 kDa.

Production and partial purification of thermostable bacteriocins from Bacillus pumilus ZED17 and DFAR8 strains with antifungal activity.

The bacteria which are members of the genus Bacillus are known to produce a wide variety of antimicrobial substances and bacteriocins. The main objective of this study was to investigate the effect of these bacteriocins on eukaryotic cells such as fungi, yeast and plant seeds. Several strains were screened for antifungal activities and identified by the means of polymerase chain reaction (PCR) of the 16s rRNA gene and sequencing. Our experiments showed that the Bacillus pumilus ZED17 and DFAR8 strains, had antifungal activities against Rhizoctonia solani and selected for further investigations. Extracellular peptides produced by these strains were purified by ammonium sulfate precipitation and dialysis. Addition of these peptides to Potato Dextrose Agar (PDA) medium inoculated with R. solani indicated significant inhibition of the fungal growth. The antifungal peptides were thermo-stable and remained active after boiling at 100˚C for 15 min. The molecular weight of the peptide with antifungal activity was estimated by electrophoresis on the Sodium Dodecyl Sulfate Poly Acrylamide Gel (SDS-PAGE) as about 5 KDa. Structural nature of this peptide was determined after gel extraction by Fourier-transform infrared spectroscopy (FTIR). Moreover, this peptide showed inhibiting effect on seeds germination of some herbs. This peptide could be applied to control herbal fungal disease induced by R. solani which is a broad host range plant pathogen fungus and its inhibition is very important. The peptide also prevents seed germination. Hence, it can be appropriate for inhibiting weeds growth. No significant effect against Saccharomyces cerevisiae and Candida albicans was observed.

Cytokines and immunoglobulin g response in donkeys with spontaneous Setaria equine infection

Setaria equina (S. equina) is a filarial worm that exists in peritoneal cavity of equines. This study aimed to evaluate cytokine mediators tumour necrosis factor alpha (TNF-α), interleukin-4 (IL-4) and immunoglobulin G (IgG) responses in spontaneously S. equina infected and non-infected donkeys with emphasis on choosing the best antigen that could be used in diagnosis of such filarial infection. A total of 87 donkeys were examined. Two S. equina antigens: crude somatic S. equina antigen (CSS) and excretory secretory S. equina antigen (ESS) were prepared. They were evaluated in diagnosis of the infection using indirect ELISA and electrophoretically characterised through sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE) and western blotting technique. The results indicated that both TNF-α and IL-4 in the serum of infected donkeys were significantly higher compared with the non-infected group at P<0.05 and P<0.01, respectively. However, the IL-4 level of infected donkeys was significantly higher than that of TNF-a (P<0.01). Apparent prevalence, specificity and positive predictive values (96.55%, 100%, and 100% each) of CSS showed higher diagnostic accuracy than that of ESS. In addition, electrophoretic protein profile and IgG reactivity of CSS antigen via western blot presented a prominent reactive protein band at 28 kDa. It was concluded that the CSS antigen was the best antigen that could be used in serodiagnosis of S. equina infection. The cytokine responses were explored in order to differentiate infected from non-infected donkeys.

Reactivation of heat-inactivated Ku proteins by heat shock cognate protein HSC73

Purpose: Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity.Methods: Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72.Results: The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37 °C after heat treatment at 44 °C for 15 min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3 h of incubation at 37 °C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3 h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment.Conclusions: These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72.

Engineering the bacterial endophyte Burkholderia pyrrocinia JK-SH007 for the control of lepidoptera larvae by introducing the cry218 genes of Bacillus thuringiensis

ABSTRACTTo expand the usage of endophytes in agriculture and in forestry, the insecticidal gene cry218 of Bacillus thuringiensis was introduced into a poplar bacterial endophyte Burkholderia pyrrocinia JK-SH007. The cry218 gene was cloned by polymerase chain reaction (PCR) and was inserted into a PHKT2 expression vector that was introduced into the bacterial endophyte JK-SH007. By using sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDS-PAGE) and western blotting, we confirmed that the engineered bacterial endophyte was successfully constructed, and it harboured insecticidal function after the bioassay in planta. The toxicity of the expressed insecticidal protein was analysed on second instar silkworm. The regression equation showed that the median lethal concentration (LC50) of the insecticidal protein was 0.77 (0.57–1.04) g/L at 72 h. The insecticidal bacteria genetically modified in this study have laid the foundation for further exploitation of biocontrol bacteria.

Poly Acrylamide Gel Electrophoresis of Serum Protein, Application to Kala-azar Patients

Sera samples were collected from 60 children aged 4-60 months, all were clinically and serologically proven cases of visceral leishmaniasis, as well as from 10 healthy children, all were seronegative with no history of parasitic infection who serve as a control during the study. Serum total protein and albumin were measured and compared between the control and visceral leishmaniasis patients. Serum protein profiles have been investigated using the conventional sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS-PAGE). Serum of control group showed the specific protein pattern with five protein bands, while serum protein profile in visceral leishmaniasis patients revealed three groups of electrophoretic banding patterns. , 50% showed twelve bands, 36.66% of the patients showed nine bands and 13.33% showed ten bands. At least four of these bands were found to be common among the infected groups which may be of diagnostic value and required further investigations. The three different electrophoretic patterns groups may be correlated with the previous epidemiological observations in respect of different clinical presentation of the disease and different response the chemotherapeutic agents at many endemic areas around the world.
 Key words: Protein band, protein patterns, serum protein, sodium dodecyl sulphate-poly acrylamide gel electrophoresis (SDS-PAGE), visceral leishmaniasis.

Urine Protein SDS-PAGE Reveals Different Profiles in Iraqi Children with Kala-azar

Urine proteomics have been an area of interest and recently in Kala-azar as an alternative sample type for serum or plasma. Because of simplicity, noninvasiveness of collection and simpler matrix. Many studies had detected an increased protein excretion in the urine of patients with active Kala-azar due to renal involvement particularly by an immunological related mechanism(s). This study have demonstrated the presence of three different protein profiles in Iraqi children (Patients: including 60 children aged 4-60 months) with defined Kala-azar using the conventional SDS-PAGE on urine samples. Urine protein profile in Kala-azar patients revealed three groups of banding patterns: group-1(33.4)% of the patients show the pattern of 5 bands with a MW. Ranged (512.861-158.489), groups-2, 3 were (55, 11.6) % of the patients showing 2 banding proteins with a MW. ranged (512.861-199.526) , (199.526-181.97) respectively. These findings may be correlated with other epidemiological studies that revealed the presence of different clinical presentations like fever, spleenomegally, hepatomegally, thrombocytopaenia, and different response to leishmania therapy. Furthermore, the presence of different protein patterns may also be related to the chronicity of infection and the degree of renal involvement. The presence of a similar protein band between group-1 and 2 may be of diagnostic purpose and further studies on expanded number of patients are required to identify that kind of protein or other urine protein profiles. 
 Key words: Protein band, protein pattern, sodium dodecyl sulphate-poly acrylamide gel electrophoresis (SDS-PAGE), urine proteome, visceral leishmaniasis.

In-silico design, expression, and purification of novel chimeric Escherichia coli O157:H7 OmpA fused to LTB protein in Escherichia coli.

E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC), and fatal hemolytic uremic syndrome (HUS) and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2–4 of E. coli O157:H7, outer membrane protein A (OmpA), and B subunit of E. coli heat labile enterotoxin (LTB) which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+) expression vector and transferred to E. coli BL21(DE3) cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3) cells with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG). The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient expression and purification of OmpA-LTB divalent under the above-mentioned conditions.