A new approach for the separation of antithrombin III with high performance affinity chromatography (HPAC) was described. A novel monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) beads (PGMA) were u...A new approach for the separation of antithrombin III with high performance affinity chromatography (HPAC) was described. A novel monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) beads (PGMA) were used as the affinity support. With the water-soluble carbodiimide, heparin was linked covalently to amino-PGMA-beads, which was prepared by amination of PGMA. The adsorbent obtained exhibits high binding activity to antithrombin III (ATIII), good resolution and excellent mechanical properties and can be used under high flow rate.展开更多
Cell membrane affinity chromatography has been widely applied in membrane protein(MP)-targeted drug screening and interaction analysis.However,in current methods,the MP sources are derived from cell lines or recombina...Cell membrane affinity chromatography has been widely applied in membrane protein(MP)-targeted drug screening and interaction analysis.However,in current methods,the MP sources are derived from cell lines or recombinant protein expression,which are time-consuming for cell culture or purification,and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase.In this study,a novel in situ synthesis membrane protein affinity chromatography(iSMAC)method was developed utilizing cell-free protein expression(CFE)and covalent immobilized affinity chromatography,which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase.The advantages of iSMAC are:1)There is no need to culture cells or prepare recombinant proteins;2)Specific and purified MPs with stable and controllable content can be obtained within 2 h;3)MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase;4)The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites.iSMAC was successfully applied to screen PDGFRβinhibitors from Salvia miltiorrhiza and Schisandra chinensis.Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h.Two new PDGFRβinhibitors,salvianolic acid B and gomisin D,were screened out with KD values of 13.44 and 7.39μmol/L,respectively.In vitro experiments confirmed that the two compounds decreasedα-SMA and collagen Ⅰ mRNA levels raised by TGF-βin HSC-T6 cells through regulating the phosphorylation of p38,AKT and ERK.In vivo,Sal B could also attenuate CCl_(4)-induced liver fibrosis by downregulating PDGFRβdownstream related protein levels.The iSMAC method can be applied to other general MPs,and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.展开更多
Glypican-3 is a protein encoded by the Glypican-3 gene located on human X chromosome (Xq26), composed of two subunits, a 40 kDa N-terminal subunit, and a 30 kDa C-terminal subunit. Glypican-3 is a currently potential ...Glypican-3 is a protein encoded by the Glypican-3 gene located on human X chromosome (Xq26), composed of two subunits, a 40 kDa N-terminal subunit, and a 30 kDa C-terminal subunit. Glypican-3 is a currently potential target molecule for liver cancer treatments because of its over-expression and growth effects on hepatocellular carcinoma (HCC). This study examined the expression and purification of a C-terminal subunit of Glypican-3 protein (GPC3-Csub) due to its application in both diagnosis and therapy for hepatocellular carcinoma. The gene encoding for GPC3-Csub was successfully cloned into plasmid pET28a fused with an affinity tag composed of six consecutive histidine residues (His-tag). Recombinant protein GPC3-Csub was expressed in Escherichia coli BL21 (DE3) in the condition of adding 3% ethanol with IPTG induction. GPC3-Csub was extracted using repeated freeze-thaw cycles with lysozyme, and inclusion bodies were solubilized by 8M Urea, SDS 10% in pH 12. His-tag fused GPC3-Csub proteins allowed it to be purified by affinity chromatography method using the Nickel-nitrilotriacetic acid (Ni-NTA) column. High expression of GPC3-Csub was confirmed by Coomassie staining and western-blot. GPC3-Csub could be isolated with a Ni-NTA column and have a purity of about 90%.展开更多
An affinity adsorbent, benzamidine Sepharose 4B, was used to separate and purify thrombin like enzymes. The p aminobenzamidine as a specific ligand was coupled to the matrix-Sepharose 4B. The recombinant thrombin like...An affinity adsorbent, benzamidine Sepharose 4B, was used to separate and purify thrombin like enzymes. The p aminobenzamidine as a specific ligand was coupled to the matrix-Sepharose 4B. The recombinant thrombin like enzyme-defibrase was used as a model in order to evaluate the efficiency of this biospecific affinity adsorbent. The homogeneity of the enzyme preparation was comfirmed as one band on sodium dodecyl sulfate polyacrylamide gel electrophoresis.展开更多
The homogeneity between the high-and low-affinity glucocorticoid receptors(GR_H and GR_L)was testified by immunoaffinity chromatography using Mab N250(recognizing the immunogenic domain at the N terminal of GR_H)and M...The homogeneity between the high-and low-affinity glucocorticoid receptors(GR_H and GR_L)was testified by immunoaffinity chromatography using Mab N250(recognizing the immunogenic domain at the N terminal of GR_H)and Mab BuGR1(recognizing the DNA binding domain of GR_H).The specific binding peak of 0.98μmol/L[~3H]triamcinolone acetonide(TA)was higher than that of 52.50nmol/L[~3H]TA.The re-sult suggests that the antigenic determinants of GR_L are similar to those of GR_H.ThecDNA of rat liver GR_H was introduced into GR_H-and GR_L-negative mouse fibroblast cellline E82.A3 by calcium phosphate coprecipitation.A number of clones which expressGR_H were selected with G418(400μg/ml).The results of radioligand binding assay indicatethat GR_H gene is expressed successfully and GR_L also may be encoded by GR_H gene.展开更多
Process analytical technology(PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance.Currently, biopha...Process analytical technology(PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance.Currently, biopharmaceutical producers mainly monitor chromatographic processes with ultraviolet/visible(UV/Vis) absorbance. However, this measurement has a very limited correlation with purity and quantity. The current study aims to determine the concentration of monoclonal antibody(mAb) and host cell proteins(HCPs) using a build-in UV/Vis monitoring during Protein A affinity chromatography and to optimize the separation conditions for high purity of mAb and minimizing the HCPs content. The eluate was analyzed through in-line UV/Vis at 280 and 410 nm, representing mAb and HCPs concentration,respectively. Each 0.1 column volume(CV) fraction of UV/Vis chromatogram peak area were calculated,and different separation conditions were then compared. The optimum conditions of mAb separation were found as 12 CV loading, elution at pH 3.5, and starting the collection at 0.5 CV point, resulting in high m Ab recovery of 95.92% and additional removal of 49.98% of HCP comparing with whole elution pool. This study concluded that UV/Vis-based in-line monitoring at 280 and 410 nm showed a high potential to optimize and real-time control Protein A affinity chromatography for mAb purification from HCPs.展开更多
The attenuation function of Dalbergia odorifera leaves on cerebral ischemia-reperfusion(I/R)is little known.The candidate targets for the Chinese herb were extracted from brain tissues through the high-affinity chroma...The attenuation function of Dalbergia odorifera leaves on cerebral ischemia-reperfusion(I/R)is little known.The candidate targets for the Chinese herb were extracted from brain tissues through the high-affinity chromatography.The molecular mechanism of D.odorifera leaves on cerebral I/R was investigated.Methods:Serial affinity chromatography based on D.odorifera leaves extract(DLE)affinity matrices were applied to find specific binding proteins in the brain tissues implemented on C57BL/6 mice by intraluminal middle cerebral artery occlusion for 1 h and reperfusion for 24 h.Specific binding proteins were subjected to mass-spectrometry to search for the differentially expressed proteins between control and DLE-affinity matrices.The hub genes were screened based on weighted gene co-expression network analysis(WGCNA).Then,predictive biology and potential experimental verification were performed for the candidate genes.The protective role of DLE in blood-brain barrier damage in cerebral I/R mice was evaluated by the leakage of Evans blue,western blotting,immunohistochemistry,and immunofluorescent staining.Results:952 differentially expressed proteins were classified into seven modules based on WGCNA under soft threshold 6.Based on WGCNA,AKT1,PIK3CA,NOS3,SMAD3,SMAD1,IL6,MAPK1,TGFBR2,TGFBR1,MAPK3,IGF1R,LRG1,mTOR,ROCK1,TGFB1,IL1B,SMAD2,and SMAD518 candidate hub proteins were involved in turquoise module.TGF-β,MAPK,focal adhesion,and adherens junction signaling pathway were associated with candidate hub proteins.Gene ontology analysis demonstrated that candidate hub proteins were related to the TGF-βreceptor signaling pathway,common-partner SMAD protein phosphorylation,etc.DLE could significantly reduce the leakage of Evans blue in mice with cerebral I/R,while attenuating the expression of occludin,claudin-5,and zonula occludens-1.Western blotting demonstrated that regulation of TGF-β/SMAD signaling pathway played an essential role in the protective effect of DLE.Conclusion:Thus,a number of candidate hub proteins were identified based on DLE affinity chromatography through WGCNA.DLE could attenuate the dysfunction of bloodbrain barrier in the TGF-β/SMAD signaling pathway induced by cerebral I/R.展开更多
[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different ...[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX.展开更多
Endotoxins(also known as lipopolysaccharides(LPS)) are undesirable by-products of recombinant proteins,purified from Escherichia coli.LPS can be considered stable under a wide range of temperature and pH,making their ...Endotoxins(also known as lipopolysaccharides(LPS)) are undesirable by-products of recombinant proteins,purified from Escherichia coli.LPS can be considered stable under a wide range of temperature and pH,making their removal one of the most difficult tasks in downstream processes during protein purification.The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration.Immobilized metal affinity chromatography(IMAC) enables the affinity interactions between the metal ions(immobilized on the support through the chelating compound) and the target molecules,thus enabling high-efficiency separation of the target molecules from other components present in a mixture.Affinity chromatography is applied with Ca2+-iminodiacetic acid(IDA) to remove most of the LPS contaminants from the end product(more than90%).In this study,the adsorption of LPS on an IDA-Ca2+ was investigated.The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal.It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads.The factors such as pH(4.0 or 5.5) and ionic strength(1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than100 EU/mL and 100 000 EU/mL.This new protocol represents a substantial advantage in time,effort,and production costs.展开更多
β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible ...β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body. After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB showed a high specific hydrolysis activity of about 1600 units /mg protein. Forty milligrams of refolded pure protein were obtained from 1L of culture.展开更多
In this study a hovel metal ion affinity ligand was immobility onto the sensor chip. Three poly-histidine peptides were used to study the interaction of tile peptides and the immobilised metal ion affinity ligand via ...In this study a hovel metal ion affinity ligand was immobility onto the sensor chip. Three poly-histidine peptides were used to study the interaction of tile peptides and the immobilised metal ion affinity ligand via biosensor system . The results obtained in this study indicate that the affinity of immobilised Ni(Ⅱ) ion affinity ligand for these peptides appear to be related to the arrangement of the histidine residues in the peptides. This study first documents the application of biosensor technique for paptide screening.展开更多
Phosphorylation plays crucial parts in lenticular biological function.Getting knowledge of region-resolved phosphoproteome contributes to better comprehending the pathogenesis.Here,we prepared the hybrid metal organic...Phosphorylation plays crucial parts in lenticular biological function.Getting knowledge of region-resolved phosphoproteome contributes to better comprehending the pathogenesis.Here,we prepared the hybrid metal organic frameworks(HMOFs)for probing the region-resolved heterogeneity of phosphoproteome in human lens.1334 phosphosites corresponding to 564 phosphoproteins,1160 phosphosites corresponding to 316 phosphoproteins and 517 phosphosites corresponding to 205 phosphoproteins were identified in capsule,cortex and nucleus,respectively,providing the relatively extensive distribution mapping of phosphorylation in human lens for the first time.The label-free quantification experiments and principal component analysis presented differential expression of phopshoproteins in three subregions.For instance,α-crystallin,β-crystallin and fibrillin-1 closely associated with cataract and Marfan syndrome showed disparate spatial distribution.The preferential phosphoproteins in capsule,cortex and nucleus were involved in cytoskeleton organization,metabolic process and lens development in camera-type eye,respectively.This work first provided a general overview of region-resolved phosphoproteome of human lens.展开更多
Food components possessing zinc ligands can be used to inhibit zinc-dependent enzymes.In this study,zinc-binding peptides were derived from whey protein hydrolysates,and their ultrafiltration(>1 and<1 kDa)fracti...Food components possessing zinc ligands can be used to inhibit zinc-dependent enzymes.In this study,zinc-binding peptides were derived from whey protein hydrolysates,and their ultrafiltration(>1 and<1 kDa)fractions,produced with Esperase(WPH-Esp),Everlase and Savinase.Immobilized metal affinity chromatography(IMAC-Zn^(2+))increased the zinc-binding capacity of the peptide fraction(83%)when compared to WPH-Esp(23%)and its<1 kDa fraction(40%).The increased zinc-binding capacity of the sample increased the inhibitory activity against the zinc-dependent“a disintegrin and metalloproteinase 17”.LC-MS/MS analysis using a shotgun peptidomics approach resulted in the identification of 24 peptides originating from bovineβ-lactoglobulin,α-lactalbumin,serum albumin,β-casein,κ-casein,osteopontin-k,and folate receptor-αin the fraction.The identified peptides contained different combinations of the strong zinc-binding group of residues,His+Cys,Asp+Glu and Phe+Tyr,although Cys residues were absent in the sequences.In silico predictions showed that the IMAC-Zn^(2+)peptides were non-toxins.However,the peptides possessed poor drug-like and pharmacokinetic properties;this was possibly due to their long chain lengths(5–19 residues).Taken together,this work provided an array of food peptide-based zinc ligands for further investigation of structure-function relationships and development of nutraceuticals against inflammatory and other zinc-related diseases.展开更多
Both glycosylation and phosphorylation exert crucial rule in multitudinous biological processes.For in-depth profiling of glycosylation and phosphorylation,a magnetic metal oxide is effectively coupled with inherently...Both glycosylation and phosphorylation exert crucial rule in multitudinous biological processes.For in-depth profiling of glycosylation and phosphorylation,a magnetic metal oxide is effectively coupled with inherently hydrophilic mesoporous channels(denoted as Fe_(3)O_(4)@TiO_(2)@mSiO_(2)-TSG).Based on the mechanism of hydrophilic interaction liquid chromatography(HILIC)and metal oxide affinity chromatography(MOAC),the Fe_(3)O_(4)@TiO_(2)@mSiO_(2)-TSG nanomaterial shows high capacity for simultaneously enriching glycopeptides and phosphopeptides.With human saliva collected in successive four days as practical biological sample,endogenous glycopeptides and phosphopeptides are efficiently enriched.Further gene ontology analysis reveals that the identified endogenous glycopeptides and phosphopeptides participate in diverse molecular functions and biological processes.This strategy is anticipated to promote variation analysis of salivary post-translational modifications.展开更多
文摘A new approach for the separation of antithrombin III with high performance affinity chromatography (HPAC) was described. A novel monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) beads (PGMA) were used as the affinity support. With the water-soluble carbodiimide, heparin was linked covalently to amino-PGMA-beads, which was prepared by amination of PGMA. The adsorbent obtained exhibits high binding activity to antithrombin III (ATIII), good resolution and excellent mechanical properties and can be used under high flow rate.
基金supported by the National Natural Science Foundation of China (Grant Nos. 82073814, 81973275, 82003909, 81973291, 82122066, 81803815)Rising-Star Program of Shanghai Science and Technology Committee (19QA1411500)
文摘Cell membrane affinity chromatography has been widely applied in membrane protein(MP)-targeted drug screening and interaction analysis.However,in current methods,the MP sources are derived from cell lines or recombinant protein expression,which are time-consuming for cell culture or purification,and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase.In this study,a novel in situ synthesis membrane protein affinity chromatography(iSMAC)method was developed utilizing cell-free protein expression(CFE)and covalent immobilized affinity chromatography,which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase.The advantages of iSMAC are:1)There is no need to culture cells or prepare recombinant proteins;2)Specific and purified MPs with stable and controllable content can be obtained within 2 h;3)MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase;4)The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites.iSMAC was successfully applied to screen PDGFRβinhibitors from Salvia miltiorrhiza and Schisandra chinensis.Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h.Two new PDGFRβinhibitors,salvianolic acid B and gomisin D,were screened out with KD values of 13.44 and 7.39μmol/L,respectively.In vitro experiments confirmed that the two compounds decreasedα-SMA and collagen Ⅰ mRNA levels raised by TGF-βin HSC-T6 cells through regulating the phosphorylation of p38,AKT and ERK.In vivo,Sal B could also attenuate CCl_(4)-induced liver fibrosis by downregulating PDGFRβdownstream related protein levels.The iSMAC method can be applied to other general MPs,and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.
文摘Glypican-3 is a protein encoded by the Glypican-3 gene located on human X chromosome (Xq26), composed of two subunits, a 40 kDa N-terminal subunit, and a 30 kDa C-terminal subunit. Glypican-3 is a currently potential target molecule for liver cancer treatments because of its over-expression and growth effects on hepatocellular carcinoma (HCC). This study examined the expression and purification of a C-terminal subunit of Glypican-3 protein (GPC3-Csub) due to its application in both diagnosis and therapy for hepatocellular carcinoma. The gene encoding for GPC3-Csub was successfully cloned into plasmid pET28a fused with an affinity tag composed of six consecutive histidine residues (His-tag). Recombinant protein GPC3-Csub was expressed in Escherichia coli BL21 (DE3) in the condition of adding 3% ethanol with IPTG induction. GPC3-Csub was extracted using repeated freeze-thaw cycles with lysozyme, and inclusion bodies were solubilized by 8M Urea, SDS 10% in pH 12. His-tag fused GPC3-Csub proteins allowed it to be purified by affinity chromatography method using the Nickel-nitrilotriacetic acid (Ni-NTA) column. High expression of GPC3-Csub was confirmed by Coomassie staining and western-blot. GPC3-Csub could be isolated with a Ni-NTA column and have a purity of about 90%.
文摘An affinity adsorbent, benzamidine Sepharose 4B, was used to separate and purify thrombin like enzymes. The p aminobenzamidine as a specific ligand was coupled to the matrix-Sepharose 4B. The recombinant thrombin like enzyme-defibrase was used as a model in order to evaluate the efficiency of this biospecific affinity adsorbent. The homogeneity of the enzyme preparation was comfirmed as one band on sodium dodecyl sulfate polyacrylamide gel electrophoresis.
文摘The homogeneity between the high-and low-affinity glucocorticoid receptors(GR_H and GR_L)was testified by immunoaffinity chromatography using Mab N250(recognizing the immunogenic domain at the N terminal of GR_H)and Mab BuGR1(recognizing the DNA binding domain of GR_H).The specific binding peak of 0.98μmol/L[~3H]triamcinolone acetonide(TA)was higher than that of 52.50nmol/L[~3H]TA.The re-sult suggests that the antigenic determinants of GR_L are similar to those of GR_H.ThecDNA of rat liver GR_H was introduced into GR_H-and GR_L-negative mouse fibroblast cellline E82.A3 by calcium phosphate coprecipitation.A number of clones which expressGR_H were selected with G418(400μg/ml).The results of radioligand binding assay indicatethat GR_H gene is expressed successfully and GR_L also may be encoded by GR_H gene.
基金supported by the National Key Research & Development Program of China (2021YFE0113300)the National Natural Science Foundation of China (22078286 and 21878263)+1 种基金Zhejiang Universitythe Talent-Introduction Program of China for the Postdoctoral Researcher for the financial support。
文摘Process analytical technology(PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance.Currently, biopharmaceutical producers mainly monitor chromatographic processes with ultraviolet/visible(UV/Vis) absorbance. However, this measurement has a very limited correlation with purity and quantity. The current study aims to determine the concentration of monoclonal antibody(mAb) and host cell proteins(HCPs) using a build-in UV/Vis monitoring during Protein A affinity chromatography and to optimize the separation conditions for high purity of mAb and minimizing the HCPs content. The eluate was analyzed through in-line UV/Vis at 280 and 410 nm, representing mAb and HCPs concentration,respectively. Each 0.1 column volume(CV) fraction of UV/Vis chromatogram peak area were calculated,and different separation conditions were then compared. The optimum conditions of mAb separation were found as 12 CV loading, elution at pH 3.5, and starting the collection at 0.5 CV point, resulting in high m Ab recovery of 95.92% and additional removal of 49.98% of HCP comparing with whole elution pool. This study concluded that UV/Vis-based in-line monitoring at 280 and 410 nm showed a high potential to optimize and real-time control Protein A affinity chromatography for mAb purification from HCPs.
基金supported by National Natural Science Foundation of China(Nos.82100417,81760094,81760724)The Foundation of Jiangxi Provincial Department of Science and Technology Project(Nos.20202ACBL206001,20212BAB206022,20181BAB205026)+1 种基金Youth Project of Jiangxi Education Department(No.GJJ200217)Open Project of Key Laboratory of Modern of TCM,Ministry of Education Jiangxi University of Traditional Chinese Medicine(TCM-2019010).
文摘The attenuation function of Dalbergia odorifera leaves on cerebral ischemia-reperfusion(I/R)is little known.The candidate targets for the Chinese herb were extracted from brain tissues through the high-affinity chromatography.The molecular mechanism of D.odorifera leaves on cerebral I/R was investigated.Methods:Serial affinity chromatography based on D.odorifera leaves extract(DLE)affinity matrices were applied to find specific binding proteins in the brain tissues implemented on C57BL/6 mice by intraluminal middle cerebral artery occlusion for 1 h and reperfusion for 24 h.Specific binding proteins were subjected to mass-spectrometry to search for the differentially expressed proteins between control and DLE-affinity matrices.The hub genes were screened based on weighted gene co-expression network analysis(WGCNA).Then,predictive biology and potential experimental verification were performed for the candidate genes.The protective role of DLE in blood-brain barrier damage in cerebral I/R mice was evaluated by the leakage of Evans blue,western blotting,immunohistochemistry,and immunofluorescent staining.Results:952 differentially expressed proteins were classified into seven modules based on WGCNA under soft threshold 6.Based on WGCNA,AKT1,PIK3CA,NOS3,SMAD3,SMAD1,IL6,MAPK1,TGFBR2,TGFBR1,MAPK3,IGF1R,LRG1,mTOR,ROCK1,TGFB1,IL1B,SMAD2,and SMAD518 candidate hub proteins were involved in turquoise module.TGF-β,MAPK,focal adhesion,and adherens junction signaling pathway were associated with candidate hub proteins.Gene ontology analysis demonstrated that candidate hub proteins were related to the TGF-βreceptor signaling pathway,common-partner SMAD protein phosphorylation,etc.DLE could significantly reduce the leakage of Evans blue in mice with cerebral I/R,while attenuating the expression of occludin,claudin-5,and zonula occludens-1.Western blotting demonstrated that regulation of TGF-β/SMAD signaling pathway played an essential role in the protective effect of DLE.Conclusion:Thus,a number of candidate hub proteins were identified based on DLE affinity chromatography through WGCNA.DLE could attenuate the dysfunction of bloodbrain barrier in the TGF-β/SMAD signaling pathway induced by cerebral I/R.
基金Supported by Anhui Provincial Natural Science Foundation of China(2008085MC65)Natural Science Foundation of Anhui Higher Education Institutions of China(KJ2021A0922)+1 种基金China Postdoctoral Science Foundation(2020T130117ZX,2020M671914)Research Activities of Postdoctoral Researchers Foundation of Anhui Province,China(2020B470)。
文摘[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX.
基金supported by grants from the Brazilian Agency Coordination of Graduate Level Training(CAPES,project 0366/09-9)State of So Paulo Research Support Foundation(FAPESP-Brazil,project 2005/60159-7)
文摘Endotoxins(also known as lipopolysaccharides(LPS)) are undesirable by-products of recombinant proteins,purified from Escherichia coli.LPS can be considered stable under a wide range of temperature and pH,making their removal one of the most difficult tasks in downstream processes during protein purification.The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration.Immobilized metal affinity chromatography(IMAC) enables the affinity interactions between the metal ions(immobilized on the support through the chelating compound) and the target molecules,thus enabling high-efficiency separation of the target molecules from other components present in a mixture.Affinity chromatography is applied with Ca2+-iminodiacetic acid(IDA) to remove most of the LPS contaminants from the end product(more than90%).In this study,the adsorption of LPS on an IDA-Ca2+ was investigated.The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal.It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads.The factors such as pH(4.0 or 5.5) and ionic strength(1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than100 EU/mL and 100 000 EU/mL.This new protocol represents a substantial advantage in time,effort,and production costs.
文摘β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body. After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB showed a high specific hydrolysis activity of about 1600 units /mg protein. Forty milligrams of refolded pure protein were obtained from 1L of culture.
文摘In this study a hovel metal ion affinity ligand was immobility onto the sensor chip. Three poly-histidine peptides were used to study the interaction of tile peptides and the immobilised metal ion affinity ligand via biosensor system . The results obtained in this study indicate that the affinity of immobilised Ni(Ⅱ) ion affinity ligand for these peptides appear to be related to the arrangement of the histidine residues in the peptides. This study first documents the application of biosensor technique for paptide screening.
基金financially supported by National Key R&D Program of China(No.2018YFA0507501)the National Natural Science Foundation of China(Nos.22074019,21425518 and 22004017)Shanghai Sailing Program(No.20YF1405300)。
文摘Phosphorylation plays crucial parts in lenticular biological function.Getting knowledge of region-resolved phosphoproteome contributes to better comprehending the pathogenesis.Here,we prepared the hybrid metal organic frameworks(HMOFs)for probing the region-resolved heterogeneity of phosphoproteome in human lens.1334 phosphosites corresponding to 564 phosphoproteins,1160 phosphosites corresponding to 316 phosphoproteins and 517 phosphosites corresponding to 205 phosphoproteins were identified in capsule,cortex and nucleus,respectively,providing the relatively extensive distribution mapping of phosphorylation in human lens for the first time.The label-free quantification experiments and principal component analysis presented differential expression of phopshoproteins in three subregions.For instance,α-crystallin,β-crystallin and fibrillin-1 closely associated with cataract and Marfan syndrome showed disparate spatial distribution.The preferential phosphoproteins in capsule,cortex and nucleus were involved in cytoskeleton organization,metabolic process and lens development in camera-type eye,respectively.This work first provided a general overview of region-resolved phosphoproteome of human lens.
基金supported by the Natural Sciences and Engineering Research Council of Canada(NSERC)Discovery Grants(RGPIN-435865-2013 and RGPIN-2018-06839)Canada Foundation for Innovation(CFI)John R.Evans Leaders Fund Infrastructure Grant(Project number:31305).
文摘Food components possessing zinc ligands can be used to inhibit zinc-dependent enzymes.In this study,zinc-binding peptides were derived from whey protein hydrolysates,and their ultrafiltration(>1 and<1 kDa)fractions,produced with Esperase(WPH-Esp),Everlase and Savinase.Immobilized metal affinity chromatography(IMAC-Zn^(2+))increased the zinc-binding capacity of the peptide fraction(83%)when compared to WPH-Esp(23%)and its<1 kDa fraction(40%).The increased zinc-binding capacity of the sample increased the inhibitory activity against the zinc-dependent“a disintegrin and metalloproteinase 17”.LC-MS/MS analysis using a shotgun peptidomics approach resulted in the identification of 24 peptides originating from bovineβ-lactoglobulin,α-lactalbumin,serum albumin,β-casein,κ-casein,osteopontin-k,and folate receptor-αin the fraction.The identified peptides contained different combinations of the strong zinc-binding group of residues,His+Cys,Asp+Glu and Phe+Tyr,although Cys residues were absent in the sequences.In silico predictions showed that the IMAC-Zn^(2+)peptides were non-toxins.However,the peptides possessed poor drug-like and pharmacokinetic properties;this was possibly due to their long chain lengths(5–19 residues).Taken together,this work provided an array of food peptide-based zinc ligands for further investigation of structure-function relationships and development of nutraceuticals against inflammatory and other zinc-related diseases.
基金supported by National Key R&D Program of China(No.2018YFA0507501)the National Science Foundation for Distinguished Young Scholars of China(No.21425518)+1 种基金the National Natural Science Foundation of China(Nos.22074019,22004017)Shanghai Sailing Program(No.20YF1405300).
文摘Both glycosylation and phosphorylation exert crucial rule in multitudinous biological processes.For in-depth profiling of glycosylation and phosphorylation,a magnetic metal oxide is effectively coupled with inherently hydrophilic mesoporous channels(denoted as Fe_(3)O_(4)@TiO_(2)@mSiO_(2)-TSG).Based on the mechanism of hydrophilic interaction liquid chromatography(HILIC)and metal oxide affinity chromatography(MOAC),the Fe_(3)O_(4)@TiO_(2)@mSiO_(2)-TSG nanomaterial shows high capacity for simultaneously enriching glycopeptides and phosphopeptides.With human saliva collected in successive four days as practical biological sample,endogenous glycopeptides and phosphopeptides are efficiently enriched.Further gene ontology analysis reveals that the identified endogenous glycopeptides and phosphopeptides participate in diverse molecular functions and biological processes.This strategy is anticipated to promote variation analysis of salivary post-translational modifications.
