1 INTRODUCTIONIn literature,most matrices of affinity chromatography for urokinase(EC 3.4.99.26)purification were prepared by cyanogen bromide activation.However,theseadsorbents usually suffered from the drawback of l...1 INTRODUCTIONIn literature,most matrices of affinity chromatography for urokinase(EC 3.4.99.26)purification were prepared by cyanogen bromide activation.However,theseadsorbents usually suffered from the drawback of leakage of the ligand,particularly inalkaline medium,because of the instability of the isourea linkage between the ligandand the spacer or agarose.Moreover,the positively charged imido group of theN-substituted isourea derivative and the hydrophobicity of the spacers might promotenonspecific adsorption.On the contrary,the adsorbents prepared by the method展开更多
Objective To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography (1MAC). Methods Phage a...Objective To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography (1MAC). Methods Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis. Results His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically. Conclusion Two specific human phage antibodies against hepatitis E virus (HEV) from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.展开更多
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
A new way for the synthesis of human interferon—α;monoclonal antibody (IFN-α;-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling...A new way for the synthesis of human interferon—α;monoclonal antibody (IFN-α;-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling efficiency and specific activity of IFN—α;-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α;(rIFN-α;) rose up to 1.03×10;IU/mg protein and the purification efficiency is appoximately 100 times.展开更多
We describe a two-step method that uses wheat germ agglutinin immobilized on Sepharose gel followed by immunoaffinity chromatography (IAC) to extract recombinant human erythropoietin and Darbepoetin from equine plasma...We describe a two-step method that uses wheat germ agglutinin immobilized on Sepharose gel followed by immunoaffinity chromatography (IAC) to extract recombinant human erythropoietin and Darbepoetin from equine plasma. Lectin affinity chromatography was shown to be an effective approach for isolating these epoetins from plasma and in combination with IAC;this method gave superior recovery when compared to the use of the latter technique alone. Moreover, due to the ease with which it can be scaled up, it is particularly well suited for pre-concentrating larger volumes of samples prior to IAC and this provides a facile way of improving the overall sensitivity with which these foreign proteins can be detected in equine plasma.展开更多
Cellulose membrane was used as Stahonary phase Of affinity chromeqraphy. A  ̄ for theprepeboon Of microPOre cellulose membone born cellulose balm is describ6d. Terne dyeS,Cibacron BIne F3GA and cave Red KZBP were immo...Cellulose membrane was used as Stahonary phase Of affinity chromeqraphy. A  ̄ for theprepeboon Of microPOre cellulose membone born cellulose balm is describ6d. Terne dyeS,Cibacron BIne F3GA and cave Red KZBP were immobilbe as amnty figeds. UP tO IO0 mgCibacron BIne F3G^ can be coupled ontO 1 g merebrane mainx. A membene cadridge cocainingblue affinty membranes was also Prepared. The flux of the cadridge was much superior to that Ofcolumn PaCked ed agarose as sepadsn odium. On this cable, the Chromatography ofhuman seam albumin was performed.展开更多
E.coli cells expressing recombinant human interferon-γ was disrupted by sonication anddissolved in 7mol·L<sup>-1</sup> guanidine hydrochloride.The extract obtained was then renaturated by 70 folddilu...E.coli cells expressing recombinant human interferon-γ was disrupted by sonication anddissolved in 7mol·L<sup>-1</sup> guanidine hydrochloride.The extract obtained was then renaturated by 70 folddilution with PBS.HulFN γ was purified by affinity chromatography with monoclonal antibody fromthe renaturated crude feed solution.After washing the column with PBS,the adsorbed HulFN γ waseluted with PBS containing 0.5mol·L<sup>-1</sup> NaCl.The column was regenerated with 2mol·L<sup>-1</sup> GuHClfor reuse.After one step of affinity purification the purity of interferon-γ was over 95%.and thespecific activity of the HulFN-γ reached 1.2×10<sup>7</sup> IU·mg<sup>-1</sup> protein.92.8% of recovery was obtainedin the elution step.Total recovery of HulFN γ activity in the affinity chromatography was 78%.展开更多
Rigid PSDVB microbeads have been modified with poly(vinyl alcohol) (PVA) adsorbed on their surface to produce an affinity medium. Then Cibacron Blue F3GA was covalently attached to the supports. The initial concentrat...Rigid PSDVB microbeads have been modified with poly(vinyl alcohol) (PVA) adsorbed on their surface to produce an affinity medium. Then Cibacron Blue F3GA was covalently attached to the supports. The initial concentration of PVA has effect on the adsorption of PVA. The non-specific interaction of bovine serum albumin (BSA) on the microbeads decreases with the PVA adsorbed. The pH stability test shows that the affinity medium is stable up to pH 11.0. And it has specific interaction with lysozyme, but not with pepsin.展开更多
A review on the principles and applications of boronic acids as affinity ligands for the chromatographic separation of carbohydrates,nucleic acid components,glycoproteins,and other small biomolecules.The mechanisms of...A review on the principles and applications of boronic acids as affinity ligands for the chromatographic separation of carbohydrates,nucleic acid components,glycoproteins,and other small biomolecules.The mechanisms of interactions between boronate ligands and analytes are described.Various boronate ligands and supports are discussed.Examples of the use of boronate affinity chromatography for separation of each class of analytes are presented.展开更多
Objective:To establish a convenient and economic method to determine hepatoma-specific α-fetoprotein(HS-AFP) for diagnosis of hepatocellular carcinoma(HCC). Methods:HS-AFP from serum of HCC patients was separated by ...Objective:To establish a convenient and economic method to determine hepatoma-specific α-fetoprotein(HS-AFP) for diagnosis of hepatocellular carcinoma(HCC). Methods:HS-AFP from serum of HCC patients was separated by a mini-column Lens culinaris agglutinin(LCA)-affinity chromatography. The levels of serum total AFP and separated HS-AFP were detected by radioimmunoassay(RIA). Results:Circulating AFP was separated into three peaks(AFP-1,AFP-2,and AFP-3) by LCA-affinity chromatography. During the elution course,the AFP-1 and AFP-2 could be eluted with TE buffer. HS-AFP(AFP-3) from sera of HCC patients was eluted clearly on the LCA-sepharose gel mini-column with a solution containing α-methyl-D-mannoside. It was a part of total AFP and only found in sera of HCC patients. A ratio of more than 15% for HS-AFP to total AFP in serum was considered as a specific marker for HCC diagnosis with higher sensitivity(92.7%) and specificity(88.2%). Conclusion:The new assay for circulating HS-AFP analysis is more sensitive,repeatable,and convenient. Its clini-cal application would be useful to early diagnosis of HCC.展开更多
A new kind of electrophoretic affinity chromatography (EAC) for bioseparation was proposed,Separation by EAC was conducted in a multicompartment electrolyzer in which the affinity gel media were packed in one of the c...A new kind of electrophoretic affinity chromatography (EAC) for bioseparation was proposed,Separation by EAC was conducted in a multicompartment electrolyzer in which the affinity gel media were packed in one of the central compartments.The presence of an electric field accelerated the migration of proteins inside the gel matrix during adsorption and descrption processes,This led to the increase of the overall speed of separation,The present study was focused on the effect of the strength of the electric field on adsorption and desorption processes.展开更多
Frontal affinity chromatography was applied to characterizing the mechanism of binding of silver with sediment particulates collected from Lake Ontario, Canada. The results showed that there was one major binding site...Frontal affinity chromatography was applied to characterizing the mechanism of binding of silver with sediment particulates collected from Lake Ontario, Canada. The results showed that there was one major binding site for Ag+ in the particulates. The binding capacity ranges from 6.06 to 1.01 μ·mol·g-1, and the binding constant (lgK) from 6.23 to 7.43 M-1 in 0.005 M ion strength at pH=3-7. The binding capacity and affinity constant were found to be pH-dependent. It is suggested that the particulate surface site where silver was bound was the anionic base. This study would be helpful for better understanding of the fundamental environmental chemistry of silver in sediments.展开更多
[Objectives]This study was conducted to purify mutton samples by gel permeation chromatography(GPC).[Methods]Fourteen organophosphorus pesticide residues in samples were qualitatively and quantitatively analyzed by ga...[Objectives]This study was conducted to purify mutton samples by gel permeation chromatography(GPC).[Methods]Fourteen organophosphorus pesticide residues in samples were qualitatively and quantitatively analyzed by gas chromatography-mass spectrometry(GC-MS)in selective ion scanning mode(SIM).[Results]The organophosphorus pesticide standard solutions showed good linearity in the mass concentration range of 0.1-10.0μg/ml with correlation coefficients(r)not lower than 0.999,and the detection limits(S=3 N)ranged from 0.01 to 0.05 mg/kg.The average recovery values were in the range of 80.2%-99.7%,with relative standard deviations(RSDs,n=3)in the range of 1.8%-6.3%,at the addition levels of 0.5,1.0 and 2.0 mg/kg.[Conclusions]The method is simple,sensitive and accurate,and can be used for the determination of organophosphorus pesticide residues in mutton.展开更多
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
A new and an inexpensive adsorbent of chitosan coated silica for immobilized metal affinity chromatography (IMIC) was studied. After a double coating, the chitosan coated on silica beads could be up to 53. 4 mg/g sili...A new and an inexpensive adsorbent of chitosan coated silica for immobilized metal affinity chromatography (IMIC) was studied. After a double coating, the chitosan coated on silica beads could be up to 53. 4 mg/g silica beads.When pH>3. 8, the metal ligand Cu2+ was chelated on the coated chitosan witha bound capacity of 14. 6 mg/g chitosan without introducing iminodiacetic acid(IDA).展开更多
Immobilized triazine dye affinity chromatography has been widely used for protein purification. In this paper, Cibacron Blue F3G-A was immobilized, through a spacer arm, onto a rigid hydrophilic porous polymer by reac...Immobilized triazine dye affinity chromatography has been widely used for protein purification. In this paper, Cibacron Blue F3G-A was immobilized, through a spacer arm, onto a rigid hydrophilic porous polymer by reacting an epoxy-group-containing poly(vinyl alcohol) with 6-aminohexyl-N(-Cibacron Blue F3G-A, which was obtained by reacting Cibacron Blue F3G-A with excess of 1,6-diaminohexane, in a pH 8.6 buffer. The epoxy-group-containing poly(vinyl alcohol) was prepared by treating macroporous poly(vinyl alcohol) with excess epichlorohydrin in the presence of NaOH in dimethyl sulfoxide. The macroporous poly(vinyl alcohol) was prepared by hydrolysis of macroporous crosslinked poly(vinyl acetate), which was synthesized by suspension copolymerization of vinyl acetate and triallyl isocyanurate in the presence of butyl acetate and n-heptane as diluents. The Cibacron Blue F3G-A-immobilized poly(vinyl alcohol) was packed in a stainless steel column (250×5 mm I. D.) and the chromatographic behaviors of several proteins (cytochrome c, lysozyme, bovine serum albumin, insulin, and lactate dehydrogenase) were determined.展开更多
A non-equilibrium chromatographic rate model was employed to simulate the affinity chromatography of urokinase. The chromatography process was developed to a yield of high purity product of urokinase from crude materi...A non-equilibrium chromatographic rate model was employed to simulate the affinity chromatography of urokinase. The chromatography process was developed to a yield of high purity product of urokinase from crude materials. The affinity gel used in the process was prepared by an epichlorohydrin-activation method using epichlorohydrin activated Sepharose 4B as a matrix and p-aminobenzamidine as a ligand. The chromatographic process were numerically simulated and analyzed with the aid of VERSE-LC computer simulator. Considering the basic principles, rate model with the back mixing in column inlet was utilized in simulating and studying the effect of the column inlet pattern on other parameters. Comparison of the simulation results with the experimental data showed that the rate model can be used to describe the affinity chromatography of urokinase in a fixed bed column with satisfactory accuracy.展开更多
Geranylgeranyl diphosphate synthase (GGPPS) plays a key role in the biosynthesis of antioxidative ca-rotenoid from the extremely radioresistant bacterium Deinococcus radiodurans. In this work, the recombinant GGPPS ex...Geranylgeranyl diphosphate synthase (GGPPS) plays a key role in the biosynthesis of antioxidative ca-rotenoid from the extremely radioresistant bacterium Deinococcus radiodurans. In this work, the recombinant GGPPS expressed in Escherichia coli by cloning and transforming the gene dr1395 of D. radiodurans was isolated rapidly by an immobilized metal affinity supermacroporous cryogel, i.e., Cu2+-iminodiacetic acid (IDA)-cryogel. The properties of the Cu2+-IDA-cryogel were characterized using capillary-based mathematical model and experi-mental measurements. The obtained protein samples were analyzed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The porosity of the present Cu2+-IDA-cryogel is 90.4% and the water permeabil-ity is 5.04×10-12 m2. From the capillary-based model, this cryogel presents a slightly wide normal pore (capillary) size distribution with the mean diameter of 55.2 μm, the standard deviation of 28.0 μm and the half of skeleton wall thickness of 2.8 μm. The pore size distribute from about 10 to 141 μm and the effective tortuosity of these capillary pores increases from 2.60 to 9.05. The isolation of the GGPPS from cell homogenate can be achieved at the flow velocity of 3.40×10-4 m·s-1 by the Cu2+-IDA-cryogel bed. High-purity GGPPS (about 91.4%) is obtained according to the SDS-PAGE analysis of the elution samples, indicating that the present method is a promising, simple and ef-fective approach to isolate GGPPS from cell homogenate of engineering strains.展开更多
基金Supported by the National Natural Science Foundation of China.
文摘1 INTRODUCTIONIn literature,most matrices of affinity chromatography for urokinase(EC 3.4.99.26)purification were prepared by cyanogen bromide activation.However,theseadsorbents usually suffered from the drawback of leakage of the ligand,particularly inalkaline medium,because of the instability of the isourea linkage between the ligandand the spacer or agarose.Moreover,the positively charged imido group of theN-substituted isourea derivative and the hydrophobicity of the spacers might promotenonspecific adsorption.On the contrary,the adsorbents prepared by the method
基金supported by the National High Technology Research and Development Program (No. 2003AA2Z3539)the Natural Science Foundation of Fujian Province (No. C0310005)the Science and Technology Foundation of Xiamen (No. 3502Z20055002).
文摘Objective To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography (1MAC). Methods Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis. Results His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically. Conclusion Two specific human phage antibodies against hepatitis E virus (HEV) from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.
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.
文摘A new way for the synthesis of human interferon—α;monoclonal antibody (IFN-α;-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling efficiency and specific activity of IFN—α;-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α;(rIFN-α;) rose up to 1.03×10;IU/mg protein and the purification efficiency is appoximately 100 times.
文摘We describe a two-step method that uses wheat germ agglutinin immobilized on Sepharose gel followed by immunoaffinity chromatography (IAC) to extract recombinant human erythropoietin and Darbepoetin from equine plasma. Lectin affinity chromatography was shown to be an effective approach for isolating these epoetins from plasma and in combination with IAC;this method gave superior recovery when compared to the use of the latter technique alone. Moreover, due to the ease with which it can be scaled up, it is particularly well suited for pre-concentrating larger volumes of samples prior to IAC and this provides a facile way of improving the overall sensitivity with which these foreign proteins can be detected in equine plasma.
文摘Cellulose membrane was used as Stahonary phase Of affinity chromeqraphy. A  ̄ for theprepeboon Of microPOre cellulose membone born cellulose balm is describ6d. Terne dyeS,Cibacron BIne F3GA and cave Red KZBP were immobilbe as amnty figeds. UP tO IO0 mgCibacron BIne F3G^ can be coupled ontO 1 g merebrane mainx. A membene cadridge cocainingblue affinty membranes was also Prepared. The flux of the cadridge was much superior to that Ofcolumn PaCked ed agarose as sepadsn odium. On this cable, the Chromatography ofhuman seam albumin was performed.
文摘E.coli cells expressing recombinant human interferon-γ was disrupted by sonication anddissolved in 7mol·L<sup>-1</sup> guanidine hydrochloride.The extract obtained was then renaturated by 70 folddilution with PBS.HulFN γ was purified by affinity chromatography with monoclonal antibody fromthe renaturated crude feed solution.After washing the column with PBS,the adsorbed HulFN γ waseluted with PBS containing 0.5mol·L<sup>-1</sup> NaCl.The column was regenerated with 2mol·L<sup>-1</sup> GuHClfor reuse.After one step of affinity purification the purity of interferon-γ was over 95%.and thespecific activity of the HulFN-γ reached 1.2×10<sup>7</sup> IU·mg<sup>-1</sup> protein.92.8% of recovery was obtainedin the elution step.Total recovery of HulFN γ activity in the affinity chromatography was 78%.
文摘Rigid PSDVB microbeads have been modified with poly(vinyl alcohol) (PVA) adsorbed on their surface to produce an affinity medium. Then Cibacron Blue F3GA was covalently attached to the supports. The initial concentration of PVA has effect on the adsorption of PVA. The non-specific interaction of bovine serum albumin (BSA) on the microbeads decreases with the PVA adsorbed. The pH stability test shows that the affinity medium is stable up to pH 11.0. And it has specific interaction with lysozyme, but not with pepsin.
文摘A review on the principles and applications of boronic acids as affinity ligands for the chromatographic separation of carbohydrates,nucleic acid components,glycoproteins,and other small biomolecules.The mechanisms of interactions between boronate ligands and analytes are described.Various boronate ligands and supports are discussed.Examples of the use of boronate affinity chromatography for separation of each class of analytes are presented.
基金grant from the Project of the Bureau of Science and Technology of Nantong (S30033).
文摘Objective:To establish a convenient and economic method to determine hepatoma-specific α-fetoprotein(HS-AFP) for diagnosis of hepatocellular carcinoma(HCC). Methods:HS-AFP from serum of HCC patients was separated by a mini-column Lens culinaris agglutinin(LCA)-affinity chromatography. The levels of serum total AFP and separated HS-AFP were detected by radioimmunoassay(RIA). Results:Circulating AFP was separated into three peaks(AFP-1,AFP-2,and AFP-3) by LCA-affinity chromatography. During the elution course,the AFP-1 and AFP-2 could be eluted with TE buffer. HS-AFP(AFP-3) from sera of HCC patients was eluted clearly on the LCA-sepharose gel mini-column with a solution containing α-methyl-D-mannoside. It was a part of total AFP and only found in sera of HCC patients. A ratio of more than 15% for HS-AFP to total AFP in serum was considered as a specific marker for HCC diagnosis with higher sensitivity(92.7%) and specificity(88.2%). Conclusion:The new assay for circulating HS-AFP analysis is more sensitive,repeatable,and convenient. Its clini-cal application would be useful to early diagnosis of HCC.
基金Supported by the State Key Projects(No.96c-03-04-05).
文摘A new kind of electrophoretic affinity chromatography (EAC) for bioseparation was proposed,Separation by EAC was conducted in a multicompartment electrolyzer in which the affinity gel media were packed in one of the central compartments.The presence of an electric field accelerated the migration of proteins inside the gel matrix during adsorption and descrption processes,This led to the increase of the overall speed of separation,The present study was focused on the effect of the strength of the electric field on adsorption and desorption processes.
基金the National Natural Science Foundation of China (Grant Nos. 40525011 and 40632011)the Chinese Academy of Sciences (kzcx2-yw-102)the International Cooperation Project of CAS for their financial support
文摘Frontal affinity chromatography was applied to characterizing the mechanism of binding of silver with sediment particulates collected from Lake Ontario, Canada. The results showed that there was one major binding site for Ag+ in the particulates. The binding capacity ranges from 6.06 to 1.01 μ·mol·g-1, and the binding constant (lgK) from 6.23 to 7.43 M-1 in 0.005 M ion strength at pH=3-7. The binding capacity and affinity constant were found to be pH-dependent. It is suggested that the particulate surface site where silver was bound was the anionic base. This study would be helpful for better understanding of the fundamental environmental chemistry of silver in sediments.
基金Supported by The Fourth Batch of High-end Talent Project in Hebei Province.
文摘[Objectives]This study was conducted to purify mutton samples by gel permeation chromatography(GPC).[Methods]Fourteen organophosphorus pesticide residues in samples were qualitatively and quantitatively analyzed by gas chromatography-mass spectrometry(GC-MS)in selective ion scanning mode(SIM).[Results]The organophosphorus pesticide standard solutions showed good linearity in the mass concentration range of 0.1-10.0μg/ml with correlation coefficients(r)not lower than 0.999,and the detection limits(S=3 N)ranged from 0.01 to 0.05 mg/kg.The average recovery values were in the range of 80.2%-99.7%,with relative standard deviations(RSDs,n=3)in the range of 1.8%-6.3%,at the addition levels of 0.5,1.0 and 2.0 mg/kg.[Conclusions]The method is simple,sensitive and accurate,and can be used for the determination of organophosphorus pesticide residues in mutton.
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A new and an inexpensive adsorbent of chitosan coated silica for immobilized metal affinity chromatography (IMIC) was studied. After a double coating, the chitosan coated on silica beads could be up to 53. 4 mg/g silica beads.When pH>3. 8, the metal ligand Cu2+ was chelated on the coated chitosan witha bound capacity of 14. 6 mg/g chitosan without introducing iminodiacetic acid(IDA).
基金The Project Grant of Science and Technology of the Education Ministry of China.
文摘Immobilized triazine dye affinity chromatography has been widely used for protein purification. In this paper, Cibacron Blue F3G-A was immobilized, through a spacer arm, onto a rigid hydrophilic porous polymer by reacting an epoxy-group-containing poly(vinyl alcohol) with 6-aminohexyl-N(-Cibacron Blue F3G-A, which was obtained by reacting Cibacron Blue F3G-A with excess of 1,6-diaminohexane, in a pH 8.6 buffer. The epoxy-group-containing poly(vinyl alcohol) was prepared by treating macroporous poly(vinyl alcohol) with excess epichlorohydrin in the presence of NaOH in dimethyl sulfoxide. The macroporous poly(vinyl alcohol) was prepared by hydrolysis of macroporous crosslinked poly(vinyl acetate), which was synthesized by suspension copolymerization of vinyl acetate and triallyl isocyanurate in the presence of butyl acetate and n-heptane as diluents. The Cibacron Blue F3G-A-immobilized poly(vinyl alcohol) was packed in a stainless steel column (250×5 mm I. D.) and the chromatographic behaviors of several proteins (cytochrome c, lysozyme, bovine serum albumin, insulin, and lactate dehydrogenase) were determined.
文摘A non-equilibrium chromatographic rate model was employed to simulate the affinity chromatography of urokinase. The chromatography process was developed to a yield of high purity product of urokinase from crude materials. The affinity gel used in the process was prepared by an epichlorohydrin-activation method using epichlorohydrin activated Sepharose 4B as a matrix and p-aminobenzamidine as a ligand. The chromatographic process were numerically simulated and analyzed with the aid of VERSE-LC computer simulator. Considering the basic principles, rate model with the back mixing in column inlet was utilized in simulating and studying the effect of the column inlet pattern on other parameters. Comparison of the simulation results with the experimental data showed that the rate model can be used to describe the affinity chromatography of urokinase in a fixed bed column with satisfactory accuracy.
基金Supported by the National Natural Science Foundation of China (30830006, 20876145, 21036005), the International Science & Technology Cooperation Program from the Ministry of Science and Technology of China (1017), the Special Fund for Agroscientific Research in the Public Interest (201103007), the Fundamental Research Funds for the Central Universities and the Natural Science Foundation of Zhejiang Province (Y4080326, Y407366).
文摘Geranylgeranyl diphosphate synthase (GGPPS) plays a key role in the biosynthesis of antioxidative ca-rotenoid from the extremely radioresistant bacterium Deinococcus radiodurans. In this work, the recombinant GGPPS expressed in Escherichia coli by cloning and transforming the gene dr1395 of D. radiodurans was isolated rapidly by an immobilized metal affinity supermacroporous cryogel, i.e., Cu2+-iminodiacetic acid (IDA)-cryogel. The properties of the Cu2+-IDA-cryogel were characterized using capillary-based mathematical model and experi-mental measurements. The obtained protein samples were analyzed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The porosity of the present Cu2+-IDA-cryogel is 90.4% and the water permeabil-ity is 5.04×10-12 m2. From the capillary-based model, this cryogel presents a slightly wide normal pore (capillary) size distribution with the mean diameter of 55.2 μm, the standard deviation of 28.0 μm and the half of skeleton wall thickness of 2.8 μm. The pore size distribute from about 10 to 141 μm and the effective tortuosity of these capillary pores increases from 2.60 to 9.05. The isolation of the GGPPS from cell homogenate can be achieved at the flow velocity of 3.40×10-4 m·s-1 by the Cu2+-IDA-cryogel bed. High-purity GGPPS (about 91.4%) is obtained according to the SDS-PAGE analysis of the elution samples, indicating that the present method is a promising, simple and ef-fective approach to isolate GGPPS from cell homogenate of engineering strains.