The synthesis route was investigated and optimized for the preparation of iminodiacetic acid-polyethylene glycol (IDA-PEG) for immobilized metal ion affinity partitioning in aqueous two-phase systems. IDA-PEG was synt...The synthesis route was investigated and optimized for the preparation of iminodiacetic acid-polyethylene glycol (IDA-PEG) for immobilized metal ion affinity partitioning in aqueous two-phase systems. IDA-PEG was synthesized from PEG in two steps by the reaction of iminodiacetic acid with a monosubstituted derivative of epichlorohydrin-activated PEG. The Cu2+ content combined with IDA-PEG was determined by atomic absorption spectrometry as 0.5 mol·mol^-1 (PEG). Furthermore, the affinity partitioning behavior of lactate dehydrogenase in polyethylene glycol/hydroxypropyl starch aqueous two-phase systems was studied to clarify the affinity effect of the Cu(Ⅱ)-IDA-PEG.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
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
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
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
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
A procedure for the preparation of continuous rod consisting of poly(glycidyl methacrylate\|co\|ethylene dimethacrylate) for immobilized metal affinity chromatography (IMAC) is presented. When chelating Cu(Ⅱ), Zn(Ⅱ)...A procedure for the preparation of continuous rod consisting of poly(glycidyl methacrylate\|co\|ethylene dimethacrylate) for immobilized metal affinity chromatography (IMAC) is presented. When chelating Cu(Ⅱ), Zn(Ⅱ) or Ni(Ⅱ) on it, the rod displayed a property of IMAC and the selectivity for protein separation was different from that obtained from the naked rod. An abnormal increase in retention times of proteins was found under the condition of either very high pH or very low pH on the metal chelated rods.展开更多
基金Supported by the National Natural Science Foundation of China(No.29736180).
文摘The synthesis route was investigated and optimized for the preparation of iminodiacetic acid-polyethylene glycol (IDA-PEG) for immobilized metal ion affinity partitioning in aqueous two-phase systems. IDA-PEG was synthesized from PEG in two steps by the reaction of iminodiacetic acid with a monosubstituted derivative of epichlorohydrin-activated PEG. The Cu2+ content combined with IDA-PEG was determined by atomic absorption spectrometry as 0.5 mol·mol^-1 (PEG). Furthermore, the affinity partitioning behavior of lactate dehydrogenase in polyethylene glycol/hydroxypropyl starch aqueous two-phase systems was studied to clarify the affinity effect of the Cu(Ⅱ)-IDA-PEG.
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).
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.
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.
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。
文摘A procedure for the preparation of continuous rod consisting of poly(glycidyl methacrylate\|co\|ethylene dimethacrylate) for immobilized metal affinity chromatography (IMAC) is presented. When chelating Cu(Ⅱ), Zn(Ⅱ) or Ni(Ⅱ) on it, the rod displayed a property of IMAC and the selectivity for protein separation was different from that obtained from the naked rod. An abnormal increase in retention times of proteins was found under the condition of either very high pH or very low pH on the metal chelated rods.
