葡聚糖接枝型高载量金属螯合介质的制备与性能

对琼脂糖凝胶微球进行烯丙基活化,再接枝葡聚糖分子,考察葡聚糖分子量等因素对葡聚糖接枝过程的影响;以葡聚糖接枝琼脂糖凝胶微球为基质,制备亚氨基二乙酸型金属螯合介质,考察葡聚糖接枝过程对金属螯合介质的孔道结构、流通性能和载量等的影响. 结果表明,分子量20~500 kDa的葡聚糖都能均匀分布于琼脂糖凝胶微球内,葡聚糖接枝量随分子量增加而增大,所制的金属螯合介质形貌、粒径及其分布基本不受影响,且具有更好的流通性能,孔道结构比商品介质Ni Sepharose 6FF更丰富. 葡聚糖接枝的金属螯合介质对带组氨酸标签的乳酸脱氢酶和睫状神经营养因子的载量分别达到19和27 mg/mL,较Ni Sepharose 6FF的载量分别提高26.6%和42.0%.     

基因重组10BNP在大肠杆菌中的表达与纯化

在摇瓶中对基因重组脑钠素（BNP）在大肠杆菌中的表达进行了研究，确定诱导后最佳培养时间为4h，表达量达21．8％，采用金属螯合亲和层析法对十聚体脑钠素进行了纯化，比较了咪唑洗脱法和降pH值洗脱法对目标蛋白的纯化效果，得到了一条纯化目标蛋白最佳亲和层析纯化路线，为后续实验打下了很好的基础。     

聚丙烯腈基螯合纤维的研究进展

回顾了聚丙烯腈基螯合纤维近年的研究进展,介绍了聚丙烯腈基螯合纤维的制备方法,螯合机理以及其应用情况,并对今后聚丙烯腈基螯合纤维的发展进行了展望。认为开发具有消臭、抗菌等多功能纤维,制备纳米金属/聚丙烯腈基复合纤维,将会被更多关注,前景乐观。     

金属螯合亲和层析在纯化重组类人胶原蛋白中的应用

研究了应用金属螯合亲和层析法纯化重组类人胶原蛋白的条件。分别考察了不同金属离子、平衡缓冲液pH值及两种洗脱方法对纯化结果的影响。结果表明,用锌离子螯合介质,pH7.5磷酸盐缓冲液平衡,上样后用100mmol·L-1氯化铵洗脱纯化效果最佳。纯化后重组类人胶原蛋白的纯度达98%,回收率为85.4%,纯化倍数为3.4。SDS-聚丙烯酰胺凝胶电泳与N端序列分析结果均证明所得纯品为重组类人胶原蛋白。     

一种新型壳聚糖分离介质的制备

以壳聚糖为载体、液体石蜡为分散介质、戊二醛为交联剂、Span80 为乳化剂,采用反相悬浮法制备壳聚糖微球,以环氧氯丙烷为活化剂、亚氨基二乙酸为螯合配基制备新型壳聚糖分离介质,并研究分离介质制备过程中各参数对壳聚糖分离介质性能的影响。确定最佳活化工艺为：40% DMSO/NaOH（0.6 mol/L）、ECH体积分数10%、反应温度40 ℃、反应时间4 h,测得环氧基修饰密度可达到0.15 mmol/g（gel）;最佳螯合工艺：IDA（0.6 mol/L）/ NaOH（2.0 mol/L）混合液,反应时间为6 h,制备的新型壳聚糖分离介质对Cu²⁺吸附量达到172.787 g/g gel,壳聚糖分离介质含水率为45.60%,孔隙率为69.45% ,得到一种新型金属螯合层析填料。     

螯合态微量元素系列肥料的制备研究

介绍了螯合态微量元素系列肥料的性质和作用，确立了最佳工艺和制备技术，并讨论了温度、酸度、螯合比等影响螯合反应的有关问题。     

改性大豆蛋白凝胶对金属离子螯合能力的研究

用乙二胺四乙酸二酐（EDTAD）对大豆蛋白进行酰化改性,再用戊二醛交联制得凝胶,研究了凝胶对Pb^2＋、Zn^2＋、Cu^2＋、Ca^2＋的螯合能力,探讨了离子浓度、温度、pH值和改性程度对凝胶金属螯合能力的影响。凝胶对Ca^2＋、Zn^2＋、Pb^2＋、Cu^2＋的最大螯合分别为0．71、0．65、0．75、0．58mmol/g。结果表明,该凝胶具有作为金属螯合剂应用于污水处理中的可能性。     

螯合型水性催干剂的应用研究

通过对不同金属催干剂的应用试验,介绍了螯合型金属催干剂的性能、优点及其在不同乳液体系中的应用结果。     

偕胺肟基螯合纤维的研究进展

介绍了偕胺肟基螯合纤维的制备方法及其与金属离子作用的螯合机理、吸附等温线、吸附反应影响因素等;概述了偕胺肟基螯合纤维在提取金属、废水处理净化等方面的应用;综述了对偕胺肟纤维—金属离子配合物应用于离子交换与吸附材料、抗菌纤维、催化剂等方面的研究;展望了偕胺肟基螯合纤维的发展趋势;指出制备力学性能较好的偕胺肟基螯合纤维的关...     

锌离子螯合亲和层析分离纯化谷胱甘肽的研究

以谷胱甘肽的酵母抽提液为原料,用自制的以壳聚糖小球为载体、Zn2 为配基的金属螯合亲和层析柱分离纯化谷胱甘肽(GSH).用含0.5 mol·L-1NH4Cl的pH值4.2、0.02 mol·L-1的磷酸盐缓冲溶液洗脱,然后经浓缩、冷冻干燥处理,即得到分离纯化的GSH.结果表明,上柱谷胱甘肽抽提液浓度为 132.30 mg·(100 mL)-1、上柱pH值为7.0时,谷胱甘肽回收率为68.61%;洗脱液中氯化铵浓度为0.5 mol·L-1、洗脱pH值为6.0时,谷胱甘肽洗脱回收率达到44.47%.     

无水体系中环氧氯丙烷活化琼脂糖凝胶制备高载量固定化金属亲和层析介质

在以二甲基亚砜为溶剂的无水体系中,利用环氧氯丙烷对琼脂糖凝胶Sepharose 6 Fast Flow进行活化,并偶联亚氨基二乙酸和Cu2+制备了固定化金属亲和层析介质. 结果表明,该体系中环氧氯丙烷对琼脂糖凝胶的活化效率大幅度提高,在40%(j)环氧氯丙烷、0.02 g/mL NaOH及50℃、反应时间4 h的优化条件下,环氧基活化密度最高达165 mmol/mL,较目前报道的最高值提高50%以上. 最终所制介质的Cu2+螯合密度为128.3 mmol/mL,对BSA的平衡吸附容量达2.05 mmol/L. 以0.5 mol/L咪唑为洗脱剂,被吸附的BSA洗脱率可达90%以上.     

Adsorption behaviors of recombinant proteins on hydroxyapatite-based immobilized metal affinity chromatographic adsorbents

The equilibrium adsorption of three homo-oligomeric model recombinant proteins containing up to 8 poly(histidine) affinity tags on a hydroxyapatite-based immobilized metal affinity chromatography (IMAC) adsorbent is reported in this study. The experimental data are well fitted with the three-parameter Langmuir-Freundlich isotherm model, indicating the presence of positive cooperativity for the adsorption of these model proteins. The maximum capacity and the binding affinity of the IMAC adsorbent for the model proteins are in principle dependent on the size and the number of affinity tags of proteins, respectively. The exceptionally high association constant of the octameric racemase, probably due to simultaneous multipoint attachment, make it difficult to elute racemase from the adsorbent. The adsorption isotherms under denaturing conditions are well fitted with the Langmuir model. Results of Scatchard analysis further suggest the homogeneous adsorption of the model protein subunits under denaturing conditions. The binding capacities and affinities of the adsorbent under denaturing conditions for the three unfolded protein subunits become essentially identical because the molecular size and number of poly(His) tags of the unfolded polypeptide chains of the three protein subunits are the same. The significant reduction in association constants under denaturing conditions suggests that high concentration of urea could interfere with the binding of proteins on the hydroxyapatite-based adsorbent.     

应用固定化金属亲和层析纯化His6融合甲状旁腺激素相关蛋白

应用原核细胞高效表达载体在大肠杆菌表达了N端带 6个组氨酸的重组人PTHrP。利用其对金属鳌合层析介质的亲合性 ,采用含盐咪唑梯度洗脱 ,一步纯化即可得到纯度大于 90 %且具有生物学活性的重组PTHrP ,并进一步制备了抗PTHrP单克隆抗体。应用His6纯化标签的固定化金属亲和层析 (IMAC) ,极大地方便了目的蛋白的纯化 ,为进一步研究PTHrP的生物学特性及其在临床诊断治疗中的应用奠定了基础。     

N-和C-末端组氨酸标记基因重组AxCeSD的柱层析分离特性

Immobilization metal affinity chromatography （IMAC）and size-exclusive chromatography （SEC）have been widely used in the purification of recombinant protein.In order to apply the column chromatography to the separation and purification of the gene recombinant with histidine-tags,the column chromatographic separation characteristics of N-terminal histidine-tagged （N-AxCeSD）and C-terminal histidine-tagged （C-AxCeSD）gene recombinant protein AxCeSD,one of the subunit involved in the cellulose synthesis in Acetobacter xylinum were studied.In the ring-shaped three-dimensional structure of AxCeSD,N-terminal histidine-tags were located in the inner of ring,while C-terminal histidine-tags were located in the outer.A higher imidazole concentration was necessary for eluting the C-AxCeSD from the IMAC column due to the C-terminal histidine-tags had stronger chelating interaction with the Ni²⁺ on the IMAC media.Moreover,the retention time for eluting C-AxCeSD from the same SEC gel column was shorter than that for N-AxCeSD,because the larger protein homolog was formed in the C-AxCeSD solution through the inter-molecular hydrogen bonds between the C-terminal histidine-tags.     

Engineering of a metal coordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinity chromatography of homologous rat enzymes

Rat glutathione transferase (GST) 3-3 binds to Ni(II)-iminodiaceticacid (IDA)-agarose, whereas other GSTs that are abundant inrat liver do not bind to this immobilized metal ion affinitychromatography (IMAC) adsorbent Rat GST 3-3 contains two superficiallylocated amino acid residues, His84 and His85, that are suitablypositioned for coordination to Ni(II)-IDA-agarose. This particularstructural motif is lacking in GSTs that do not bind to theIMAC matrix. Creation of an equivalent His-His structure inthe homologous human GST M1-1 by protein engineering affordeda mutant enzyme that displays affinity for Ni(II)-IDA-agarose,in contrast to the wild-type GST M1-1. The results identifya distinct site that is operational in IMAC and suggest an approachto the rational design of novel integral metal coordinationsites in proteins.     

A poly(2-hydroxyethyl methacrylate)-based immobilized metal affinity chromatography adsorbent for protein purification

Poly(HEMA) microbeads were prepared by suspension polymerization of 2-hydorxyethylmethacrylate and ethyleneglycoldimethacrylate (EGDMA). The water content, ligand density, and selectivity for poly(His)-tagged d-hydantoinase of the poly(HEMA)-based adsorbents were affected by the concentration of EDGMA used during polymerization. The Ni(II)-loaded poly(HEMA) adsorbent exhibited an adsorption capacity of 1.0 mg/g for poly(His)-tagged d-hydantoinase under optimal conditions with buffer containing 100-300 mM NaCl at pH 6.0. One-step purification protocol with the adsorbent gave a purity of at least 92%. The adsorption capacity of adsorbent declined by 54% after 7 cycles, due to the leaching of Ni(II) from the adsorbent. However, upon regeneration the adsorption capacity can be restored. Given the ease of preparation and the chemical and microbial resistance, the poly(HEMA)-based IMAC adsorbent could be a promising substitute for the polysaccharide-based IMAC adsorbents.     

pH值和盐浓度对金属螯合亲和层析分离人胰高血糖素样肽-1融合蛋白的影响

目的探讨pH值和盐浓度对金属螯合亲和层析(Immobilized metal ion affinity chromatography,IMAC)分离含His-Tag标签的人胰高血糖素样肽-1(Glucagon-like peptide-1,GLP-1)融合蛋白的影响,并确定最佳洗脱条件。方法在0.50mol/L盐浓度条件下,分别取pH6.0、7.0、7.8和9.04种缓冲液进行咪唑梯度洗脱;在合适的pH值条件下,分别取氯化钠浓度0.25、0.50和0.75mol/L3种缓冲液进行咪唑梯度洗脱。分析在不同pH值及盐浓度条件下洗脱融合蛋白所需要的咪唑浓度及回收率。结果在所考察的pH值范围内,洗脱融合蛋白所需的咪唑浓度随洗脱液pH值的升高而逐渐降低,当洗脱液pH值为7.8时,融合蛋白的分离效果最佳,大部分杂蛋白被0～40mmol/L咪唑洗脱液去除且不含融合蛋白,而绝大部分融合蛋白在咪唑浓度达80～200mmol/L时被洗出,总回收率达(83.7±1.0)%,且比例较高;在此pH值条件下,氯化钠浓度达0.75mol/L,才会影响融合蛋白的回收率,氯化钠浓度为0.25或0.50mol/L的洗脱液洗出融合蛋白的效果相近;在pH7.8,含0.25mol/L氯化钠的洗脱体系下,用50mmol/L咪唑溶液洗脱杂蛋白,200mmol/L咪唑溶液洗脱融合蛋白,融合蛋白的回收率和比例分别可达(85.2±2.0)%和(80.5±1.0)%。结论在所考察的pH值和盐浓度范围内,提高洗脱液pH值和盐浓度均可降低洗脱融合蛋白需要的咪唑量,但也会降低融合蛋白的回收率。