Our previous studies have reported the presence of "chain delivery" effects of protein adsorption onto ion exchangers with polymer-grafted ion-exchange groups, such as dextran-grafted and poly(ethylenimine)-...Our previous studies have reported the presence of "chain delivery" effects of protein adsorption onto ion exchangers with polymer-grafted ion-exchange groups, such as dextran-grafted and poly(ethylenimine)-modified Sepharose gels. However, it is unclear if the "chain delivery" occurs on affinity adsorption with specific interactions. This work is designed to address this issue. A dextran-grafted Sepharose gel was prepared, and then the matrix was modified using diethylaminoethyl, a typical ion-exchange group, or octapeptide(FYCHWQDE), an affinity ligand for human immunoglobulin G(h Ig G) to prepare ion-exchange or affinity adsorbents, respectively.Results of h Ig G adsorption showed that the uptake rate represented by the effective diffusivity of h Ig G onto the dextran-grafted ion exchangers was obviously enhanced by the dextran grafting, indicating the presence of"chain delivery" of the bound proteins on the charged groups on the dextran chains. By contrast, the effective diffusivity of h Ig G changed little as ligand density increased on the dextran-grafted FYCHWQDE adsorbents.Their adsorption capacities decreased and effective diffusivities were not accelerated by the dextran grafting.Thus, this work clarified that grafted dextran could not accelerate h Ig G uptake rate on the affinity resins, or in other words, chain delivery did not occur on the specific interaction-based affinity adsorption.展开更多
Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of cAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by dis-playing it...Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of cAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by dis-playing it on the surface of filamentous bacteriophage fd. Phosphorylation in vitro by cAPK showed a unique labelled band of approximately 60 ku, which was consistent with the molecular weight of the PKS-g3p fusion protein. Some weakly phosphorylated bands for both PKS phage and wild-type phage were also observed. Phage display random 15-mer peptide library phosphorylated by cAPK was selected with ferric (Fe3+ ) chelalion affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. The results showed that 5 of 14 sequenced phages displayed the cAPK recognition sequence motif (R)RXS/T. Their in vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typical (R)RXS/T sequence motif. It suggested that the new method of using ferric (Fe 3+ ) chelation affinity chromatography to identify the substrate specificity of protein kinase from random peptide library was feasible.展开更多
在大肠杆菌中表达的重组Hepcidin融合蛋白以包涵体形式存在,其N端带有6个组氨酸。以Ni2+-IDA-Sepharose Fast Flow为层析介质,在变性条件下以不同的咪唑和pH值洗脱方式对Hepcidin融合蛋白的纯化效果进行了比较,确定了该融合蛋白的金属...在大肠杆菌中表达的重组Hepcidin融合蛋白以包涵体形式存在,其N端带有6个组氨酸。以Ni2+-IDA-Sepharose Fast Flow为层析介质,在变性条件下以不同的咪唑和pH值洗脱方式对Hepcidin融合蛋白的纯化效果进行了比较,确定了该融合蛋白的金属螯合层析纯化条件。以60 mmol/L咪唑洗脱杂蛋白,然后将pH值降至4.0洗脱融合蛋白,纯化后的融合蛋白纯度大于95%,而且不含咪唑,有利于下一步Hepcidin的制备。金属螯合层析中融合蛋白收率不低于90%。Ni2+-IDA-Sepharose Fast Flow对该融合蛋白的吸附量为30.4 mg/mL。展开更多
基金Supported by the National Natural Science Foundation of China(21236005,21621004)
文摘Our previous studies have reported the presence of "chain delivery" effects of protein adsorption onto ion exchangers with polymer-grafted ion-exchange groups, such as dextran-grafted and poly(ethylenimine)-modified Sepharose gels. However, it is unclear if the "chain delivery" occurs on affinity adsorption with specific interactions. This work is designed to address this issue. A dextran-grafted Sepharose gel was prepared, and then the matrix was modified using diethylaminoethyl, a typical ion-exchange group, or octapeptide(FYCHWQDE), an affinity ligand for human immunoglobulin G(h Ig G) to prepare ion-exchange or affinity adsorbents, respectively.Results of h Ig G adsorption showed that the uptake rate represented by the effective diffusivity of h Ig G onto the dextran-grafted ion exchangers was obviously enhanced by the dextran grafting, indicating the presence of"chain delivery" of the bound proteins on the charged groups on the dextran chains. By contrast, the effective diffusivity of h Ig G changed little as ligand density increased on the dextran-grafted FYCHWQDE adsorbents.Their adsorption capacities decreased and effective diffusivities were not accelerated by the dextran grafting.Thus, this work clarified that grafted dextran could not accelerate h Ig G uptake rate on the affinity resins, or in other words, chain delivery did not occur on the specific interaction-based affinity adsorption.
文摘Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of cAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by dis-playing it on the surface of filamentous bacteriophage fd. Phosphorylation in vitro by cAPK showed a unique labelled band of approximately 60 ku, which was consistent with the molecular weight of the PKS-g3p fusion protein. Some weakly phosphorylated bands for both PKS phage and wild-type phage were also observed. Phage display random 15-mer peptide library phosphorylated by cAPK was selected with ferric (Fe3+ ) chelalion affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. The results showed that 5 of 14 sequenced phages displayed the cAPK recognition sequence motif (R)RXS/T. Their in vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typical (R)RXS/T sequence motif. It suggested that the new method of using ferric (Fe 3+ ) chelation affinity chromatography to identify the substrate specificity of protein kinase from random peptide library was feasible.
文摘在大肠杆菌中表达的重组Hepcidin融合蛋白以包涵体形式存在,其N端带有6个组氨酸。以Ni2+-IDA-Sepharose Fast Flow为层析介质,在变性条件下以不同的咪唑和pH值洗脱方式对Hepcidin融合蛋白的纯化效果进行了比较,确定了该融合蛋白的金属螯合层析纯化条件。以60 mmol/L咪唑洗脱杂蛋白,然后将pH值降至4.0洗脱融合蛋白,纯化后的融合蛋白纯度大于95%,而且不含咪唑,有利于下一步Hepcidin的制备。金属螯合层析中融合蛋白收率不低于90%。Ni2+-IDA-Sepharose Fast Flow对该融合蛋白的吸附量为30.4 mg/mL。
