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Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.
Subject(s)
Humans , Bacterial Proteins , Diphtheria Toxin/genetics , Escherichia coli/genetics , Molecular Chaperones/genetics , Recombinant Proteins/geneticsABSTRACT
Objective::Astragalus membranaceus var. mongholicus and A. membranaceus are medicinal Astragalus, which are closely related and similar in composition, but with unclear medicinal value. Water-soluble protein profiles for A. membranaceus var. mongholicus and A. membranaceus were established to explore the differences between the two kinds of Astragalus Radix. Method::The water-soluble protein components were obtained through water ultrasonic extraction and acetone precipitation. After digested with trypsin, the obtained peptides were analyzed by nano ESI-LC-MS/MS method. Proteome Discovery 1.4 software was used to identify the proteins by comparing with the legume protein database, and the different expression water-soluble proteins were analyzed by the label-free quantitative software SIEVE. Finally, relevant information for common expression proteins, including classification, molecular function, involved biological process and signaling pathway, were analyzed by bioinformatics. Result::There were 920 and 717 specific proteins identified for A. membranaceus var. mongholicus and A. membranaceus, respectively. Totally 472 proteins were found to be co-expressed, in which 21 were differentially expressed, such as PR-10 protein, NDK-1 protein, glutelin A2, and phospholipase D. There were 14 highly expressed proteins in A. membranaceus var. mongholicus and 7 highly expressed proteins in A. membranaceus. Conclusion::There are significant differences in water-soluble protein profiles for two kinds of Astragalus Radix. Specific proteins, differentially expressed proteins and common expressed proteins can provide references for the identification of A. membranaceus var. mongholicus and A. membranaceus. It also can be used to define pharmacological mechanisms and search for drug action targets.
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Objective To analyze and evaluate the chemical components from flowers of Astragalus membranaceus var. mongholicus, and to investigate the potential value of the medicinal plant resources. Methods UV-Vis spectrophotometry was used to determine the total contents of polysaccharides and water-soluble protein. HPLC-PDA/ELSD method was used to determine monosaccharides and oligosaccharides and GC-MS method was used to determine volatile components and the fatty acids in the flowers of A. membranaceus var. mongholicus. UPLC-TQ-MS method was used to analyze the nucleosides and amino acids. Results The flowers of A. membranaceus var. mongholicus contain abundant polysaccharides (47.02 mg/g), water-soluble protein (470.66 mg/g), fructose (45.46 mg/g), glucose (8.71 mg/g), and sucrose (1.05 mg/g). There were 32 kinds of volatile components detected in the flowers of A. membranaceus var. mongholicus, in which oxy-derivatives were the main components. In addition, six nucleosides and 15 amino acids were detected in the flowers of A. membranaceus var. mongholicus, and their total contents were 2.77 mg/g and 6.52 mg/g, respectively. Eight fatty acids in the flowers of A. membranaceus var. mongholicus were also detected, in which myristic acid, palmitic acid, and oleic acid were the main components. Conclusion This study investigated the composition and content of various nutritional components of the flower of A. membranaceus var. mongholicus, which provides a scientific basis for its utilization and development.
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Objective To investigate the anti-inflammatory effect of the protein derived from the soluble factor of Heligmosomoides polygyrus (H. polygyrus) excretory-secretory in a colitis model. Methods Colitis was induced by providing drinking water containing 3% dextran sodium sulfate (DSS) for a week. DSS was administrated in a cycle protocol, each cycle consisted of 7 days of 3% DSS in the drinking water and followed by 7 days of regular water. This study consisted of five treatment groups, including Groups A (control) received untreated water, B (DSS only, without excretory-secretory), and C–E injected (i.p.) with excretory-secretory protein (H. polygyrus excretory-secretory total, excretory-secretory 28 kDa and excretory-secretory 55 kDa, respectively). Mice received injection every week. The injection of excretory-secretory was started from the 6th weeks and continued until 11 weeks. At the end of 11 weeks of the experiment, mice were sacrificed, colon tissue was removed and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, flow cytometry, real-time PCR and histology examination. Results Mice received H. polygyrus excretory-secretory 55 kDa reduced mono-nuclear cell infiltrations. H. polygyrus excretory-secretory 55 kDa induced the down-regulation of mRNA interferon-γ expression. There were significant differences in the expression of mRNA interferon in the colon of mice after the administration of the excretory-secretory 55 kDa protein fraction compared with other groups (P < 0.001), whereas mRNA transforming growth factor-β expression up regulated in the colon of mice after the administration of the excretory-secretory 55 kDa protein fraction compared with total excretory-secretory group (P < 0.05). The treatment of colitis in mice with excretory-secretory 55 kDa protein fractions modulated interleukin-10 (IL-10) expression, whereas excretory-secretory total and excretory-secretory 28 kDa protein fractions insufficient promoted IL-10 expression. Excretory-secretory 55 kDa proteins fraction promoted IL-10 expression via Foxp3-independent pathways. Conclusions Excretory-secretory 55 kDa protein could reduce inflammation and have potential therapy. H. polygyrus excretory-secretory 55 kDa was the soluble factor that may help in the development of novel treatments to cure colitis.
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Objective: To investigate the anti-inflammatory effect of the protein derived from the soluble factor of Heligmosomoides polygyrus (H. polygyrus) excretory-secretory in a colitis model. Methods: Colitis was induced by providing drinking water containing 3% dextran so-dium sulfate (DSS) for a week. DSS was administrated in a cycle protocol, each cycle consisted of 7 days of 3%DSS in the drinking water and followed by 7 days of regular water. This study consisted of five treatment groups, including Groups A (control) received untreated water, B (DSS only, without excretory-secretory), and C–E injected (i.p.) with excretory-secretory protein (H. polygyrus excretory-secretory total, excretory-secretory 28 kDa and excretory-secretory 55 kDa, respectively). Mice received injection every week. The injection of excretory-secretory was started from the 6th weeks and continued until 11 weeks. At the end of 11 weeks of the experiment, mice were sacrificed, colon tissue was removed and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, flow cytometry, real-time PCR and histology examination. Results: Mice received H. polygyrus excretory-secretory 55 kDa reduced mono-nuclear cell infiltrations. H. polygyrus excretory-secretory 55 kDa induced the down-regulation of mRNA interferon-g expression. There were significant differences in the expression of mRNA interferon in the colon of mice after the administration of the excretory-secretory 55 kDa protein fraction compared with other groups (P Conclusions: Excretory-secretory 55 kDa protein could reduce inflammation and have potential therapy. H. polygyrus excretory-secretory 55 kDa was the soluble factor that may help in the development of novel treatments to cure colitis.
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Objective To express and purify the glycoprotein extracellular domain (Ex-GP) of Rabies virus strain CTN in soluble form with high efficiency.Methods A recombinant expression plasmid containing the gene encoding the Ex-GP was constructed.Various expression conditions were screened to obtain an optimum prokaryotic expression system for Ex-GP in soluble form.The expressed target protein was purified using affinity chromatography and gel filtration chromatography.Results The target protein Ex-GP with high antigenicity was efficiently expressed in soluble form by using the recombinant PBCX expression system and effectively purified by using affinity and gel filtration chromatography.Conclusion The soluble form of Ex-GP is successfully expressed and purified in a simple and convenient way.This study paves the way for further researches on the biological functions of rabies virus glycoprotein,the pathogenic mechanism of rabies and the development of diagnostic reagent and vaccines for rabies virus.
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Objective To study the effect of purslane decoction on oxidative stress products and soluble protein of senile cataract patients tears.Methods 120 cases of cataract patients (160 eyes) were selected, according to different drugs were randomly assigned to two groups.The control group were treated with Imidacloprid sinnock sodium drops eye drops, 1 drop each time, 4 times per day,the patients in the experimental group on the basis of the control group with Portulaca oleracea decoction 100 mL, for three consecutive months.SOD, MDA, soluble protein level and visual improvement were compared after the end of treatment.Results Compared with control group, the visual acuity of the experimental group was higher(P<0.05), MDA was lower, SOD was higher(P<0.05), soluble protein content was higher(P<0.05).Conclusion Portulaca oleracea decoction can apparently improve the cataract patients with tear of SOD, MDA, soluble protein level and improve visual acuity.
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Objective To express and purify the envelope ( E) protein of Japanese encephalitis virus (JEV) in soluble form.Methods Various prokaryotic expression vectors , host strains and induction conditions including time and temperatures were screened to obtain an optimum prokaryotic expression system for JEV E protein in soluble form .The expressed protein was purified by using nickel column chromatography and gel filtration chromatography .Results The soluble JEV E protein , accounting for 23% of the totally bacteria soluble protein was effectively expressed by using the recombinant plasmid PBCX -E406 at low tem-perature.The purity of the expressed protein reached up to 85%after the purification by using nickel column and gel filtration chromatography .Conclusion Soluble JEV E protein was successfully expressed and puri-fied in a simple and efficient way .It would provide a useful tool for further investigation on JEV infection , attenuation mechanism of JE live vaccine strain SA 14-14-2 and the quality control of JE vaccine .It can also be used for the development of diagnosis assay for JEV .
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In the present study, in vitro mutagenesis techniques were applied to investigate the effects of gamma irradiation at 0, 10, 20, 30, 40, 50 and 60 Gy on physiological changes in callus of Gerbera jamesonii Bolus ex. Hook f. Biochemical changes in chlorophyll and soluble protein content of pre- and post- irradiated Gerbera callus were studied. Non-irradiated callus demonstrated the highest amount of chlorophyll content as compared to callus irradiated at 10, 20, 30, 40, 50 and 60 Gy. In addition, the amount of chlorophyll b was relatively higher than chlorophyll a in both the irradiated and non-irradiated callus, except for callus irradiated at 10 Gy. Biochemical differentiation based on total soluble protein content revealed gradual reduction after day 9 of exposure to gamma irradiation. Reduction of soluble protein content was observed in all the treatments as the increase of incubation period.
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Feather waste is generated in large amounts as a by-product of commercial poultry processing. This residue is almost pure keratin, which is not easily degradable by common proteolytic enzymes. Eight strains of Bacillus, isolated from decomposing feathers were tested for the hydrolysis of feather wastes in the laboratory. Among these strains, Bacillus cereus KB043 was the best feather degrading organism when grown on basal medium containing 1 percent hen feather as sole source of carbon and nitrogen. It caused 78.16 ± 0.4 percent degradation with a significant release of soluble protein (1206.15 ± 14.7 µg mL-1) and cysteine (20.63 ± 0.4 µg mL-1) in the cultivation fluid. The strain also showed the highest level of keratinase activity (39.10 ± 0.4 U mL-1). These data indicates that the Bacillus cereus KB043 could be useful in management of poultry wastes.
Subject(s)
Animals , Bacillus cereus , Bacillus/enzymology , Bacillus/isolation & purification , Enzyme Activation , Peptide Hydrolases/analysis , Feathers/enzymology , Keratins/analysis , Biodegradation, Environmental , Birds , MethodsABSTRACT
Objective To study the stability of the Bradford method for determination of soluble protein in animal materials. Method The absorption of CBB G-250 combined with proteins was determined by the UV-Spectrophotometry method with the detection wavelength at 595 nm,and the dried bufo was selected as sample,BSA as control. Results The method showed a good linear relationship between absorption and protein content (r =0.996). The sample was stable with in 10 min (RSD=5.0%). Conclusion This method was reliable,simple and rapid.
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Objective: To identify 5 specimens of C.tubulosa(Schenk) R.Wight on different parasitifers,and explore the relationship between electrophoretic fingerprint and identification on soluble-protein,meanwhile providing data for establishing electrophoresis pattern library of Chinese crude drugs.Methods: The electrophoretic characteristics of soluble-proteins from 5 specimens of C.tubulosa(Schenk) R.Wight on different parasitifers were studied with SDS-PAGE.Results: It indicated that reproducible and high resolvable electrophoresis patterns of soluble proteins of C.tubulosa(Schenk) R.Wight on different parasitifers by SDS-PAGE.Their degrees of polymorphism were 55.56%.Conclusion: Each specimen had its own unique band pattern distinguish from the others.The electrophoresis pattern of SDS-PAGE of soluble proteins can be regarded as protein fingerprints for identification of C.tubulosa(Schenk) R.Wight on different parasitifers.
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Objective:To investigate the effect of local application of the water soluble protein fraction,containing specific egg yolk antibody,on the recolonization of Streptococcus mutans.Methods:Whole cells of inactivated streptococcus mutans were used as antigen to immunize bens,then water soluble protein fraction(WSF) was extracted from the eggs. Mouth wash containing 0.1 mg/ml of WSF was prepared and administered to 8 volunteers.The mouth wash was used once every two days for 2 weeks.Vehicle solution was used in other 6 volunteers as the control.S.mutans in saliva was monitored for 100 days.Results:Before using the mouth washes,S.mutans level in saliva of the volunteers was 36.4%.S.mutans was removed by hibitane,it kept less than 3% in 100 days in the tested individuals,while 23%～37% in the controls.Conclusion:The WSF containing specific egg yolk antibody can effectively prevent the recolonization of streptococcus mutans.
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Objective To compare the effects of two crushing methods on dissolution rate and decocting rate of water-soluble protein in Plastrum Testudinis.Methods The dissolution rate of water-soluble protein was compared in Plastrum Testudinis processed by the traditional crushing method(passing 100-eyes screen,fine powder)and ultra-fine crushing method(passing 300-eye screen,ultra-fine powder);besides,orthogonal design was applied to study the process technical condition of decocting rate in Plastrum Testudinis.Results Water-soluble protein in fine powder of Plastrum Testudinis was hardly detected while the dissolution rate of water-soluble protein in ultra-fine powder was 51.2 %in 20 minutes and up to 70.5 %at 2 hours.Three influencing factors of fineness,decocting frequencies and decocting time were measured with orthogonal test.The results of variance analysis showed that fineness significantly influenced the decocting rate(P .05).Conclusion The ultra-fine powder technique is propitious to the increase of dissolution rate and decocting rate of water-soluble protein in Plastrum Testudinis.