The affinity heptapeptide(HWWWPAS)for insulin,selected from phage display library,was coupled to EAH Sepharose 4B gel and packed to a 1-mL column.The column was used for the affinity purification of insulin from prote...The affinity heptapeptide(HWWWPAS)for insulin,selected from phage display library,was coupled to EAH Sepharose 4B gel and packed to a 1-mL column.The column was used for the affinity purification of insulin from protein mixture and commercial insulin preparation.It was observed that the minor impurity in the commercial insulin was removed by the affinity chromatography.Nearly 40 mg of insulin could be purified with the 1-mL affinity column.The results revealed the high specificity and capacity of the affinity column for insulin purification.Moreover,based on the analysis of the amino acids in the peptide sequence,shorter peptides were designed and synthesized for insulin chromatography.As a result,HWWPS was found to be a good alternative to HWWWPAS,while the other two peptides with three or four amino acids showed weak affinity for insulin.The results indicated that the peptide sequence of HWWWPAS was quite conservative for specific binding of insulin.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
Human serum albumin （HSA） is an abundant protein in plasma that can bind and transport many small molecules, and the corresponding affinity-controlled drug delivery shows great advantage in the biological system. Pe...Human serum albumin （HSA） is an abundant protein in plasma that can bind and transport many small molecules, and the corresponding affinity-controlled drug delivery shows great advantage in the biological system. Peptide SA06 is a reported ligand comprising 20 amino acids, and is known to non-covalently bind with HSA to extend the lifetime and improve the pharmacokinetic performance. The structural information of the HSA-peptide complex is keen for obtainingmolecular insight of the binding mechanism. We studied the secondary structural change and structure-affinity relations of Peptide SA06 with HSA by using circular dichroism （CD） spectroscopy in solution. Noticeable allosteric effect can be identified by compositional increase of a-helix structures when the peptide was co-incubated with HSA. Furthermore, the equilibrium dissociation constant of Peptide SA06 with HSA can be determined by CD-baged method. This work provides structural evidence on the allosteric interaction between peptide ligand and HSA, and sheds light on optimization of therapeutic properties in the affinity-controlled delivery systems.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
Specific and dynamic biological interactions pave the blueprint of signal networks in cell. For example, a great variety of specific protein-ligand interactions define how intracellular signals flow. Taking advantage ...Specific and dynamic biological interactions pave the blueprint of signal networks in cell. For example, a great variety of specific protein-ligand interactions define how intracellular signals flow. Taking advantage of the specificity of these interactions, we postulate an "affinity-guided covalent conjugation" strategy to lock binding ligands through covalent reactions between the ligand and the receptor protein. The presence of a nucleophile close to the ligand binding site of a protein is sine qua none of this reaction. Specific noncovalent interaction of a ligand derivative(which contains an electrophile at a designed position) to the ligand binding site of the protein brings the electrophile to the close proximity of the nucleophile. Subsequently, a conjugation reaction spontaneously takes place between the nucleophile and the electrophile, and leads to an intermolecular covalent linkage. This strategy was first showcased in coiled coil peptides which include a cysteine mutation at a selected position. The short peptide sequence was used for covalent labeling of cell surface receptors. The same strategy was then used to guide the design of a set of protein Lego bricks for covalent assembly of protein complexes of unnatural geometry. We finally made "reactive peptides" for natural adaptor proteins that play significant roles in signal transduction. The peptides were designed to react with a single domain of the multidomain adaptor protein, delivered into the cytosol of neurons, and re-directed the intracellular signal of neuronal migration. The trilogy of protein labeling, assembly, and inhibition of intracellular signals, all through a specific covalent bond, fully demonstrated the generality and versatility of "affinity-guided covalent conjugation" in various applications.展开更多
目的:利用噬菌体7肽库进行体外快速差减筛选(biopanning and rapid analysis of selective interactive ligands,BRASIL),从而获得卵巢癌细胞株HO-8910细胞表面特异性结合肽。方法:以中国仓鼠卵巢细胞株CHO为吸附细胞,卵巢癌细胞株HO-8...目的:利用噬菌体7肽库进行体外快速差减筛选(biopanning and rapid analysis of selective interactive ligands,BRASIL),从而获得卵巢癌细胞株HO-8910细胞表面特异性结合肽。方法:以中国仓鼠卵巢细胞株CHO为吸附细胞,卵巢癌细胞株HO-8910为靶细胞,利用噬菌体展示技术联合差减筛选策略,经过连续5轮生物淘洗,随机挑选13个噬菌体单克隆进行DNA测序,利用噬菌体竞争结合实验、ELISA实验、细胞免疫染色、细胞免疫荧光、合成多肽的竞争抑制实验鉴定阳性噬菌体的亲和性及特异性。结果:筛选出的噬菌体NPMIRRQ与卵巢癌细胞株HO-8910有较高的亲和性及特异性。结论:阳性噬菌体表面展示的多肽NPMIRRQ可能为卵巢癌的早期诊断和转移复发监测提供新思路。展开更多
A new method for the rapid and efficient screening of affinity ligands to biological targets is reported. The fusion peptide of influenza virus A was used as the model target and immobilized on the PGMA beads. Antisen...A new method for the rapid and efficient screening of affinity ligands to biological targets is reported. The fusion peptide of influenza virus A was used as the model target and immobilized on the PGMA beads. Antisense peptide YRSKQA of fusion peptide was chosen as the lead compound. The special positional scanning peptide libraries were designed based on YRSKQA and synthesized by utilizing solid phase peptide synthesis manually. The libraries were YRSKQX, YRSKXA, YRSXQA, YRXKQA, YXSKQA and XRSKQA, where X represented 18 L-amino acids(except for Cys and Trp). Each library was screened by affinity chromatography. The eluates from the fusion peptide affinity column were collected and analyzed by RP-HPLC and MS, respectively, in order to determine the kind of X at each position. After the preferred residues of six positions were decided, the two preferred peptide sequences, GRGKHK and TRGKHK, were obtained. The dissociation constants of GRGKHK, TRGKHK and YRSKQA, were 3.35×10 -6, 5.24×10 -6 and 1.15×10 -5 mol·L -1, respectively. The preferred peptides showed the higher affinity binding to immobilized fusion peptide than the lead peptide.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
基金Supported by Natural Science Foundation of China(No.20476081)the Programfor Changjiang ScholarsInnovative Research Team in University from the Ministry of Education of China.
文摘The affinity heptapeptide(HWWWPAS)for insulin,selected from phage display library,was coupled to EAH Sepharose 4B gel and packed to a 1-mL column.The column was used for the affinity purification of insulin from protein mixture and commercial insulin preparation.It was observed that the minor impurity in the commercial insulin was removed by the affinity chromatography.Nearly 40 mg of insulin could be purified with the 1-mL affinity column.The results revealed the high specificity and capacity of the affinity column for insulin purification.Moreover,based on the analysis of the amino acids in the peptide sequence,shorter peptides were designed and synthesized for insulin chromatography.As a result,HWWPS was found to be a good alternative to HWWWPAS,while the other two peptides with three or four amino acids showed weak affinity for insulin.The results indicated that the peptide sequence of HWWWPAS was quite conservative for specific binding of insulin.
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Acknowledgement This work was supported by the National Natural Science Foundation of China （Nos. 21273051, 21673055）. The financial supports fi＇om the CAS key Laboratory of Standardization and Measurement for Nanotechnology, and the CAS key Laboratory for Biological Effects of Nanomaterials and Nanosafety are also gratefully acknowledged.
文摘Human serum albumin （HSA） is an abundant protein in plasma that can bind and transport many small molecules, and the corresponding affinity-controlled drug delivery shows great advantage in the biological system. Peptide SA06 is a reported ligand comprising 20 amino acids, and is known to non-covalently bind with HSA to extend the lifetime and improve the pharmacokinetic performance. The structural information of the HSA-peptide complex is keen for obtainingmolecular insight of the binding mechanism. We studied the secondary structural change and structure-affinity relations of Peptide SA06 with HSA by using circular dichroism （CD） spectroscopy in solution. Noticeable allosteric effect can be identified by compositional increase of a-helix structures when the peptide was co-incubated with HSA. Furthermore, the equilibrium dissociation constant of Peptide SA06 with HSA can be determined by CD-baged method. This work provides structural evidence on the allosteric interaction between peptide ligand and HSA, and sheds light on optimization of therapeutic properties in the affinity-controlled delivery systems.
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.
基金supported by the University Grants Committee of Hong Kong (ECS grant CUHK 404812, GRF grants 403711 and 404413, and Ao E/M-09/12)
文摘Specific and dynamic biological interactions pave the blueprint of signal networks in cell. For example, a great variety of specific protein-ligand interactions define how intracellular signals flow. Taking advantage of the specificity of these interactions, we postulate an "affinity-guided covalent conjugation" strategy to lock binding ligands through covalent reactions between the ligand and the receptor protein. The presence of a nucleophile close to the ligand binding site of a protein is sine qua none of this reaction. Specific noncovalent interaction of a ligand derivative(which contains an electrophile at a designed position) to the ligand binding site of the protein brings the electrophile to the close proximity of the nucleophile. Subsequently, a conjugation reaction spontaneously takes place between the nucleophile and the electrophile, and leads to an intermolecular covalent linkage. This strategy was first showcased in coiled coil peptides which include a cysteine mutation at a selected position. The short peptide sequence was used for covalent labeling of cell surface receptors. The same strategy was then used to guide the design of a set of protein Lego bricks for covalent assembly of protein complexes of unnatural geometry. We finally made "reactive peptides" for natural adaptor proteins that play significant roles in signal transduction. The peptides were designed to react with a single domain of the multidomain adaptor protein, delivered into the cytosol of neurons, and re-directed the intracellular signal of neuronal migration. The trilogy of protein labeling, assembly, and inhibition of intracellular signals, all through a specific covalent bond, fully demonstrated the generality and versatility of "affinity-guided covalent conjugation" in various applications.
文摘目的:利用噬菌体7肽库进行体外快速差减筛选(biopanning and rapid analysis of selective interactive ligands,BRASIL),从而获得卵巢癌细胞株HO-8910细胞表面特异性结合肽。方法:以中国仓鼠卵巢细胞株CHO为吸附细胞,卵巢癌细胞株HO-8910为靶细胞,利用噬菌体展示技术联合差减筛选策略,经过连续5轮生物淘洗,随机挑选13个噬菌体单克隆进行DNA测序,利用噬菌体竞争结合实验、ELISA实验、细胞免疫染色、细胞免疫荧光、合成多肽的竞争抑制实验鉴定阳性噬菌体的亲和性及特异性。结果:筛选出的噬菌体NPMIRRQ与卵巢癌细胞株HO-8910有较高的亲和性及特异性。结论:阳性噬菌体表面展示的多肽NPMIRRQ可能为卵巢癌的早期诊断和转移复发监测提供新思路。
文摘A new method for the rapid and efficient screening of affinity ligands to biological targets is reported. The fusion peptide of influenza virus A was used as the model target and immobilized on the PGMA beads. Antisense peptide YRSKQA of fusion peptide was chosen as the lead compound. The special positional scanning peptide libraries were designed based on YRSKQA and synthesized by utilizing solid phase peptide synthesis manually. The libraries were YRSKQX, YRSKXA, YRSXQA, YRXKQA, YXSKQA and XRSKQA, where X represented 18 L-amino acids(except for Cys and Trp). Each library was screened by affinity chromatography. The eluates from the fusion peptide affinity column were collected and analyzed by RP-HPLC and MS, respectively, in order to determine the kind of X at each position. After the preferred residues of six positions were decided, the two preferred peptide sequences, GRGKHK and TRGKHK, were obtained. The dissociation constants of GRGKHK, TRGKHK and YRSKQA, were 3.35×10 -6, 5.24×10 -6 and 1.15×10 -5 mol·L -1, respectively. The preferred peptides showed the higher affinity binding to immobilized fusion peptide than the lead peptide.
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.
