A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-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_A monoclonal antibody (IFN-α_A-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_A-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_A (rIFN-α_A) rose up to 1.03×10~7IU/mg protein and the purification efficiency is appoximately 100 times.展开更多
The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatogra...The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatography (SAX) in the presence of low concentration- of urea. The effect of urea concentration on this refolding process was investigated. The obtained refolded rhG-CSF has a high specific activity of 2.3×108 U/mg, demonstrating that the proteins were completely refolded during the chromatographic process. With only one step by SAX in 40 min, purity and mass recovery of the refolded and purified rhG-CSF were 97% and 43%, respectively.展开更多
Thr extract of E. containing recombinant human interferon- (rhIFN-) with 7.0 mol/L guanidine hydrochloride (Gu . HCl) was directly injected into a column of reverse phase liquid chromatography (RPLC) to separate and p...Thr extract of E. containing recombinant human interferon- (rhIFN-) with 7.0 mol/L guanidine hydrochloride (Gu . HCl) was directly injected into a column of reverse phase liquid chromatography (RPLC) to separate and purify rhIFN- with acetic acid-water as mobile phase. Gu I-ICI and most impure proteins can be separated by this way. Compared with the usual dilution method, the bioactivity recovery of the purified rhIFN- was found to be over 500%. In addition, compared to common organic solvents employed ill RPLC, acetic acid has higher freezing point, and therefore, it is easy to concentrate the aim-protein by freeze-drying when acetic acid-water is used as mobile phase ill RPLC.展开更多
The synthesis of packing of oxygen-ethane as end-ligand bonded to silica andseparation for recombinant human interferons(rhIFN) in high-performance hydrophobicinteraction chromatography has been studied in this paper....The synthesis of packing of oxygen-ethane as end-ligand bonded to silica andseparation for recombinant human interferons(rhIFN) in high-performance hydrophobicinteraction chromatography has been studied in this paper. It was showed that the packingsynthesized was not only suitable for separating standard proteins, but also for different rhIFNs,i. e., rhIFN-αA expressed in yeast, rhIFN-αA and rh1FN-γ in E. colt. The purify of threerhIFNs purified by one-step HPHIC, which rhIFN-γ expressed in E. colt was recognized as80%, rhIFN-αA expressed in yeast as 70%, and rhIFN-αA expressed in E. colt as 30%, wasmore than that of other chromatography besides affinity chromatography.展开更多
对利用金属离子亲和层析纯化重组类人胶原蛋白过程中使用的金属离子进行了比较,从而对分离纯化的条件进行优化。在相同实验条件下,用4种金属离子柱分离纯化目的蛋白。结果显示,经4种金属离子柱纯化后镍柱的总蛋白收获率最高,铜柱与锌柱...对利用金属离子亲和层析纯化重组类人胶原蛋白过程中使用的金属离子进行了比较,从而对分离纯化的条件进行优化。在相同实验条件下,用4种金属离子柱分离纯化目的蛋白。结果显示,经4种金属离子柱纯化后镍柱的总蛋白收获率最高,铜柱与锌柱居中,钙柱最低;而柱保留时间则为锌柱最高(12.38 m in),钙柱最低(8.25 m in);钙柱洗脱时所需咪唑解离初始浓度最低(100 mmol/L),锌柱最高(200 mmol/L);对SDS-PAGE电泳图进行分析得纯化后类人胶原蛋白的纯度分别为:镍柱82.8%,铜柱83.4%,锌柱96.2%,钙柱94.3%。由此可见锌柱对目标蛋白的亲和力最高,且纯化后类人胶原蛋白的纯度也最高。因此,确定锌离子作为亲和层析纯化重组类人胶原蛋白的金属离子。展开更多
A downstream purification procedure for recombinant human interferon-γ(rhIFN-γ) expressed in E. coli was described. An essentially two-step chromatographic purification procedure, i. e., size exclusion chromatograph...A downstream purification procedure for recombinant human interferon-γ(rhIFN-γ) expressed in E. coli was described. An essentially two-step chromatographic purification procedure, i. e., size exclusion chromatography(SEC) and high-performance hydrophobic in-teraction chromatography (HPHIC), was used for purification of homogeneity of rhIFN-γ from the inclusion body. The specific activity of purified rhIFN-Y was 1.0×108 IU/mg pro-tein. The product of purification rhIFN-γ was analyzed by an analytical high-per formance SEC and a significant sigle symmetrical peak had been found. The purity of purified rhIFN-Y was greater than 95% from analysis, determination by analytical HPSEC and SDS-PAGE with Coomssie Blue. The molecular weight was ca. 15 000. It was shown that this procedure was an effective method for purifying rhIFN-γ.展开更多
文摘A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-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_A-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_A (rIFN-α_A) rose up to 1.03×10~7IU/mg protein and the purification efficiency is appoximately 100 times.
基金This work is supported by the National Natural Science Foundation of China(No．20175016)
文摘The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatography (SAX) in the presence of low concentration- of urea. The effect of urea concentration on this refolding process was investigated. The obtained refolded rhG-CSF has a high specific activity of 2.3×108 U/mg, demonstrating that the proteins were completely refolded during the chromatographic process. With only one step by SAX in 40 min, purity and mass recovery of the refolded and purified rhG-CSF were 97% and 43%, respectively.
基金This work was supported by the National Natural Science Foundation of China!(29675017,39880003)
文摘Thr extract of E. containing recombinant human interferon- (rhIFN-) with 7.0 mol/L guanidine hydrochloride (Gu . HCl) was directly injected into a column of reverse phase liquid chromatography (RPLC) to separate and purify rhIFN- with acetic acid-water as mobile phase. Gu I-ICI and most impure proteins can be separated by this way. Compared with the usual dilution method, the bioactivity recovery of the purified rhIFN- was found to be over 500%. In addition, compared to common organic solvents employed ill RPLC, acetic acid has higher freezing point, and therefore, it is easy to concentrate the aim-protein by freeze-drying when acetic acid-water is used as mobile phase ill RPLC.
文摘The synthesis of packing of oxygen-ethane as end-ligand bonded to silica andseparation for recombinant human interferons(rhIFN) in high-performance hydrophobicinteraction chromatography has been studied in this paper. It was showed that the packingsynthesized was not only suitable for separating standard proteins, but also for different rhIFNs,i. e., rhIFN-αA expressed in yeast, rhIFN-αA and rh1FN-γ in E. colt. The purify of threerhIFNs purified by one-step HPHIC, which rhIFN-γ expressed in E. colt was recognized as80%, rhIFN-αA expressed in yeast as 70%, and rhIFN-αA expressed in E. colt as 30%, wasmore than that of other chromatography besides affinity chromatography.
文摘对利用金属离子亲和层析纯化重组类人胶原蛋白过程中使用的金属离子进行了比较,从而对分离纯化的条件进行优化。在相同实验条件下,用4种金属离子柱分离纯化目的蛋白。结果显示,经4种金属离子柱纯化后镍柱的总蛋白收获率最高,铜柱与锌柱居中,钙柱最低;而柱保留时间则为锌柱最高(12.38 m in),钙柱最低(8.25 m in);钙柱洗脱时所需咪唑解离初始浓度最低(100 mmol/L),锌柱最高(200 mmol/L);对SDS-PAGE电泳图进行分析得纯化后类人胶原蛋白的纯度分别为:镍柱82.8%,铜柱83.4%,锌柱96.2%,钙柱94.3%。由此可见锌柱对目标蛋白的亲和力最高,且纯化后类人胶原蛋白的纯度也最高。因此,确定锌离子作为亲和层析纯化重组类人胶原蛋白的金属离子。
文摘A downstream purification procedure for recombinant human interferon-γ(rhIFN-γ) expressed in E. coli was described. An essentially two-step chromatographic purification procedure, i. e., size exclusion chromatography(SEC) and high-performance hydrophobic in-teraction chromatography (HPHIC), was used for purification of homogeneity of rhIFN-γ from the inclusion body. The specific activity of purified rhIFN-Y was 1.0×108 IU/mg pro-tein. The product of purification rhIFN-γ was analyzed by an analytical high-per formance SEC and a significant sigle symmetrical peak had been found. The purity of purified rhIFN-Y was greater than 95% from analysis, determination by analytical HPSEC and SDS-PAGE with Coomssie Blue. The molecular weight was ca. 15 000. It was shown that this procedure was an effective method for purifying rhIFN-γ.
