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Polyhydroxyalkanoate depolymerase (PHAD) can be used for the degradation and recovery of polyhydroxyalkanoate (PHA). In order to develop a PHAD with good stability under high temperature, PHAD from Thermomonospora umbrina (TumPHAD) was heterelogously expressed in Escherichia coli BL21(DE3). At the same time, a mutant A190C/V240C with enhanced stability was obtained via rational design of disulfide bonds. Characterization of enzymatic properties showed that the mutant A190C/V240C had an optimum temperature of 60 ℃, which was 20 ℃ higher than that of the wild type. The half-life at 50 ℃ was 7 hours, at 50 ℃ which was 21 times longer than that of the wild type. The mutant A190C/V240C was used for the degradation of polyhydroxybutyrate (PHB), one of the typical PHA. At 50 ℃, the degradation rate of PHB being treated for 2 hours and 12 hours was 2.1 times and 3.8 times higher than that of the wild type, respectively. The TumPHAD mutant A190C/V240C obtained in this study shows tolerance to high temperature resistance, good thermal stability and strong PHB degradation ability, which may facilitate the degradation and recovery of PHB.
Subject(s)
Thermomonospora , Actinomycetales , Escherichia coli/genetics , PolyhydroxyalkanoatesABSTRACT
Pro-gastrin-releasing peptide (ProGRP) is a specific marker of small cell lung cancer.
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A commercial albumin-bound paclitaxel nano-formulation has been considered a gold standard against breast cancer. However, its application still restricted unfavorable pharmacokinetics and the immunogenicity of exogenous albumin carrier. Herein, we report an albumin-bound tumor redox-responsive paclitaxel prodrugs nano-delivery strategy. Using diverse linkages (thioether bond and disulfide bond), paclitaxel (PTX) was conjugated with an albumin-binding maleimide (MAL) functional group. These pure PTX prodrugs could self-assemble to form uniform and spherical nanoparticles (NPs) in aqueous solution without any excipients. By immediately binding to blood circulating albumin after intravenous administration, NPs are rapidly disintegrated into small prodrug/albumin nanoaggregates
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The formation of most proteins consists of two steps: the synthesis of precursor proteins and the synthesis of functional proteins. In these processes, propeptides play important roles in assisting protein folding or inhibiting its activity. As an important polypeptide chain coded by a gene sequence in lipase gene, propeptide usually functions as an intramolecular chaperone, assisting enzyme molecule folding. Meanwhile, some specific sites on propeptide such as glycosylated sites, have important effect on the activity, stability in extreme environment, methanol resistance and the substrate specificity of the lipase. Studying the mechanism of propeptide-mediated protein folding, as well as the influence of propeptide on lipases, will allow to regulate lipase by alternating the propeptide folding behavior and in turn pave new ways for protein engineering research.
Subject(s)
Lipase/metabolism , Molecular Chaperones/metabolism , Protein Folding , Protein Precursors , Substrate SpecificityABSTRACT
Disulfide-bond A oxidoreductase-like protein (DsbA-L) is a molecular chaperone involved in the multimerization of adiponectin. Recent studies have found that DsbA-L is related to metabolic diseases including gestational diabetes mellitus (GDM), and can be regulated by peroxisome proliferator-activated receptor γ (PPARγ) agonists; the specific mechanism, however, is uncertain. Furthermore, the relationship between DsbA-L and the novel adipokine chemerin is also unclear. This article aims to investigate the role of DsbA-L in the improvement of insulin resistance by PPARγ agonists in trophoblast cells cultured by the high-glucose simulation of GDM placenta. Immunohistochemistry and western blot were used to detect differences between GDM patients and normal pregnant women in DsbA-L expression in the adipose tissue. The western blot technique was performed to verify the relationship between PPARγ agonists and DsbA-L, and to explore changes in key molecules of the insulin signaling pathway, as well as the effect of chemerin on DsbA-L. Results showed that DsbA-L was significantly downregulated in the adipose tissue of GDM patients. Both PPARγ agonists and chemerin could upregulate the level of DsbA-L. Silencing DsbA-L affected the function of rosiglitazone to promote the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB)/AKT pathway. Therefore, it is plausible to speculate that DsbA-L is essential in the environment of PPARγ agonists for raising insulin sensitivity. Overall, we further clarified the mechanism by which PPARγ agonists improve insulin resistance.
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OBJECTIVE: To establish a mass spectrometry method for the qualitative analysis of anti-CTLA4 monoclonal antibody and its N-linked glycosylation. METHODS The anti-CTLA4 monoclonal antibody was characterized by liquid-mass technique from the aspects of intact molecular weight, subunit molecular weight, amino acid sequence coverage, disulfide bond, N-linked glycosylation site and glycoform. RESULTS: The molecular weight of anti-CTLA4 mAb (A2G0F/A2G0F) is 147 992; the deglycosylation molecular weight of anti-CTLA4 mAb is 145 103; the molecular weight of light chain is 23 450; the molecular weight of heavy chain (A2G0F) is 50 548; the heavy chain Fd segment has a molecular weight of 25 355 and the heavy chain sFc segment (A2G0F,1xK_Loss) has a molecular weight of 25 189. Peptide mapping was performed with Trypsin & Chymotrypsin, and the coverage of the amino acid sequence was 100%. The peptide containing the N-linked glycosylations site is EEQYNSTYR, and the N-glycosylation site is located at Asn298 of the heavy chains. Thirteen glycoform were characterized including A2G0F(59.8%), A2G1Fa(18.66%), A2G1Fb(7.28%), A2G2F(3.2%), M5(1.66%), A2S1G0F(1.17%), A1G0F (1.16%), A3G1F(0.95%), A2G0(0.94%), A2S2F(0.78%), A2S1G1F(0.67%), A3G0F(0.53%)and A1G1F(0.51%). CONCLUSION: A method for qualitative and quantitative analysis of monoclonal antibody is established.
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Disulfide-bond A oxidoreductase-like protein (DsbA-L) is a molecular chaperone involved in the multimerization of adiponectin. Recent studies have found that DsbA-L is related to metabolic diseases including gestational diabetes mellitus (GDM), and can be regulated by peroxisome proliferator-activated receptor γ (PPARγ) agonists; the specific mechanism, however, is uncertain. Furthermore, the relationship between DsbA-L and the novel adipokine chemerin is also unclear. This article aims to investigate the role of DsbA-L in the improvement of insulin resistance by PPARγ agonists in trophoblast cells cultured by the high-glucose simulation of GDM placenta. Immunohistochemistry and western blot were used to detect differences between GDM patients and normal pregnant women in DsbA-L expression in the adipose tissue. The western blot technique was performed to verify the relationship between PPARγ agonists and DsbA-L, and to explore changes in key molecules of the insulin signaling pathway, as well as the effect of chemerin on DsbA-L. Results showed that DsbA-L was significantly downregulated in the adipose tissue of GDM patients. Both PPARγ agonists and chemerin could upregulate the level of DsbA-L. Silencing DsbA-L affected the function of rosiglitazone to promote the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB)/AKT pathway. Therefore, it is plausible to speculate that DsbA-L is essential in the environment of PPARγ agonists for raising insulin sensitivity. Overall, we further clarified the mechanism by which PPARγ agonists improve insulin resistance.
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OBJECTIVE@#To establish a micellar electrokinetic capillary chromatography-based method for identification and quantitative detection of interleukin-12 (IL-12) and analysis of its unfolding process.@*METHODS@#An uncoated fused-silica capillary (inner diameter 50 μm) with a total length of 48.5 cm (40 cm to the detector) was used for the experiment. The factors influencing the separation efficiency of IL-12 were analyzed, and a standard curve of IL-12 concentration was established. The mixture of IL-12 and anti-IL-12 antibody was incubated in a water bath at 38 ℃ for 40 min, and capillary electrophoresis was then performed under the same conditions. The results were compared with those of IL-12 and anti-IL-12 antibody to identify IL-12. IL-12 and dithiothreitol (DTT) were incubated at 60 ℃ in water bath for different lengths of times, and the unfolding process of IL-12 was analyzed based on electrophoresis results of IL-12 in different states.@*RESULTS@#A micellar capillary electrophoresis on-line sweep method was established with 80 mmol/L borate (pH=9.3) containing 30 mmol/L sodium dodecyl sulfate (SDS) as the buffer solution. This system showed a good linear relationship between the peak area and the mass concentration of IL-12 with a linear correlation coefficient of 0.9991 within the linear range of 2 to 120 ng/L. As the incubation time of IL-12 and DTT prolonged, the disulfide bond of IL-12 gradually opened and resulted in distinct changes in the protein peak.@*CONCLUSIONS@#This capillary electrophoresis-based method is simple and sensitive for IL-2 analysis and allows rapid detection of changes in IL-12 content in the setting of tumors and analysis of the possible causes.
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Background: Xylanases are considered one of the most important enzymes in many industries. However, their low thermostability hampers their applications in feed pelleting, pulp bleaching, and so on. The main aim of this work was to improve the thermostability of Trichoderma ressei xylanase 2 (Xyn2) by introducing disulfide bonds between the N-terminal and α-helix and the ß-sheet core. Results: In this work, two disulfide bonds were separately introduced in the Xyn2 to connect the N-terminal and α-helix to the ß-sheet core of Xyn2. The two disulfide bonds were introduced by site-directed mutagenesis of the corresponding residues. The half-life of the mutants Xyn2C1452 (disulfide bond between ß-sheets B2 and B3) and Xyn2C59149 (disulfide bond between ß-sheets A5 and A6) at 60°C was improved by approximately 2.5- and 1.8-fold compared to that of the wild type Xyn2. In addition, the enzyme's resistance to alkali and acid was enhanced. Conclusion: Our results indicated that the connection of the N-terminal and α-helix to the ß-sheet core is due to the stable structure of the entire protein.
Subject(s)
Trichoderma/enzymology , Xylosidases/metabolism , Disulfides/metabolism , Mass Spectrometry , Temperature , Trichoderma/genetics , Trichoderma/metabolism , Xylans/metabolism , Xylosidases/genetics , Enzyme Stability , Kinetics , Mutagenesis, Site-Directed , Hydrogen-Ion Concentration , MutationABSTRACT
OBJECTIVE: To investigate antagonistic activities of three isomers of α-conotoxin TxIB on rat and human α6 /α3β2β3 nicotinic acetylcholine receptors (nAChRs). METHODS: Three disulfide bond isomers were synthesized using Fmoc chemistry, which were identified by ultra performance liquid chromatography (UPLC)and confirmed by MALDI-TOF mass spectrometry. Rat and human α6/α3β2β3 nAChRs were expressed in oocytes of Xenopus laevis, which were used to test the antagonistic abilities of the 3 isomers. RESULTS: The three isomers of α-conotoxin TxIB were synthesized successfully. The retention time of each isomer of α-conotoxin TxIB was different each other significantly. The observed molecular masses of three isomers were the same, which were consistent with their theoretical molecular mass. Their hydrophilicity orders were globular > ribbon > bead. Both rat and human α6/α3β2β3 nAChRs were expressed in oocytes well. Inhibition of three isomers of α-conotoxin TxIB on rat and human α6 /α3β2β3 nAChRs were evaluated respectively. Among the three isomers of TxIB, the activity of the globular isomer was the most potent one, which had almost same activity at rat and human α6/α3β2β3 nAChRs with corresponding IC50 of 28.2 and 32.0 nmol·L-1 respectively. However, the other two isomers, ribbon and bead isomers displayed little antagonistic effect on both rat and human α6/α3β2β3 nAChRs only with an IC50 of > 10 μmol·L-1. CONCLUSION: The synthesized globular isomer of α-conotoxin TxIB in this work has a high selectivity and potent antagonistic activity on rat and human α6/α3β2β3 nAChRs, which would be helpful for its new drug development.
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OBJECTIVE: To evaluate the reference standard of recombinant human interleukin-15 (IL-15) for the effective quality control of IL-15 products according to the requirements of Chinese Pharmacopeia. METHODS: The biological activity, concentration, purity, and isoelectric point of IL-15 were tested according to Chinese Pharmacopeia (volume III, 2010 edition). The primary structure was confirmed by analyzing the N-terminal amino acid sequence and relative molecular mass and peptide mass mapping. RESULTS: The measured biological activity of IL-15 was 1.03×107 IU·mg-1, the content was (0.879±0.065) mg·mL-1, the purities tested by SDS-PAGE and SEC-HPLC were all 100%, and the isoelectric point was 5.2, which all met the criteria specified in the quality standard. The observed relative molecular mass, 12 900.80, was consistent with theoretical value (12 900.71). Its amino acid sequence was verified with coverage of 100%. The disulfide bonds were identified to to be Cys36-Cys86/Cys43-Cys89, which was in accordance with the published papers. CONCLUSION: This reference standard, which is qualified and has correct amino acid sequence, can be used for the routine quality control of IL-15.
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OBJECTIVE: To characterize and compare the primary structures of recombinant human erythropoietin marketed in China. METHODS: Recombinant human erythropoietin reference substances were obtained from nine manufacturers, for which the molecular mass, peptide mass mapping, oligosaccharide profile and content of sialic acid were analyzed and compared. RESULTS: The measured molecular masses of de-N-glycosylated EPO were all in agreement with the theoretical values with mass error less than 1. All the samples had consistent amino acid sequence, disulfide bonds (Cys7-Cys161, Cys29-Cys33), O-glycosylation site (Ser126), and N-glycosylation sites (Asn24, Asn38, Asn83), but different glycosylation pattern and ratio of glycoforms. The sialic acid content of the nine samples were within 11.5-15.7 mol∶mol EPO. CONCLUSION: Recombinant human erythropoietins from nine manufacturers have identical primary structures except for glycosylation patterns.
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@#To investigate activities of three isomers of α-conotoxin TxID on human α3β4 and α6/α3β4 nicotinic acetylcholine receptors(nAChRs). The three isomers of α-conotoxin TxID were synthesized using solid phase Fmoc chemistry and fully folded by two-step oxidations. Human α3β4 and α6/α3β4 nAChRs were expressed in oocytes of Xenopus laevis, which were used for bioassay of the three isomers, including inhibition and washout reversibility. There were obvious differences between the inhibition potency of each isomers at human α3β4 and α6/α3β4 nAChRs. The blocking was reversible and washout rapidly. The most potent isomer is the globular form with an IC50 of 9. 3 nmol/L on human α3β4 and α6/α3β4 nAChRs respectively. The 2nd potent isomer was the ribbon form with much less potency, which had an IC50 of > 5 μmol/L. The bead isomer had little or no block on human α3β4 and α6/α3β4 nAChRs with an IC50 of > 10 μmol/L. The three isomers of α-conotoxin TxID were synthesized successfully with two pairs of desired disulfide bond. Inhibition activities of the 3 isomers on human α3β4 and α6/α3β4 nAChRs were obtained respectively, which would be basis for new marine drug development of α-conotoxin TxID.
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OBJECTIVE: To identify several kinds of post-translational modifications in recombinant protein pharmaceuticals by MS/MS. METHODS: Firstly the proteins were digested with enzymes, and the obtained peptides were analyzed by UPLC-MS. By MS analysis, peptides with post-translational modifications could be detected, then it was then identified by MS/MS. RESULTS: Using MS/MS, the disulfide bonds, peptides with similar molecular mass, O-glycosylation site and the type of N-linked oligosaccharides were identified. CONCLUSION: MS/MS analvsis can make up for the deficiency of enzyme digestion in some extent, and provide more sufficient evidence for the identification of post-translational modifications.
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OBJECTIVE: To introduce the application of reduction-sensitive polymeric micelles in the field of tumor targeting drug delivery. METHODS: In the past decades, tumor targeted drug delivery systems have become important research area because they promise to resolve several key therapeutical issues including low treatment efficacy and significant side effects. On the basis of published literatures, the recent developments in reduction-sensitive polymeric micelles used for tumor targeting drug delivery were reviewed, with an emphasis on their structure characteristics as well as their biomedical applications from the cellular level and animal level, respectively. RESULTS AND CONCLUSION: As a novel intelligent drug delivery system, reduction-sensitive polymeric micelles can effectively control drug release with low side effects and high therapeutic effects both in vitro and in vivo. It has great potential in tumor targeting drug delivery.
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so as to increase inclusion body by 32%when compared with placebo.Conclusion As a disulfide bond isomerase,DsbG may also have chaperone activity during EB infecting to host cell.
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The plysical and chemical characteristics of recombinant human GM - CSF (rhGM - CSF) were studied separatly. rhGM - CSF was treated by GdHCl, reduced by DTT, and the disulfide bond was blocked by idoacetamide. The results showed that the samples aren' t homogeneous in UV absorption spectrum and RP-HPLC analysis after treatment by DTT. There was no remarkable differences in the results of the analysis of SDS-PAGE electrophoresis and western blot between treated and untreated rhGM - CSF samples. The effect of GdHCl on GM-CSF was reversible in all above tests; In peptide mapping analysis, the digestion of the samples with blocked disulfide bond by CNBr and protease is more complete than that without any treatment.