离子交换法纯化重组类人胶原蛋白II的研究

用阳离子交换树脂CM-52分离纯化类人胶原蛋白II(HumanCollagen-likeBioproteinII,HCBII)。通过实验,分别确定了静态吸附和柱层析吸附的操作条件,即静态吸附:在pH4.0、NaCl0.15mol/L、进料浓度7g/L,处理量17.26ml/g树脂的条件下进行吸附,吸附时间为60min;柱层析:在pH4.0、NaCl0.15mol/L、进料浓度5g/L、流速5ml/min的条件下进行吸附。然后上柱,在pH4.0、NaCl0.30mol/L下进行脱附。实验表明,采用静态吸附的方式吸附HCBII,然后上柱洗脱,HCBII的吸附量可达到48.6mg/g树脂,回收率83.8%,操作时间短,分辨率高,最终纯化的HCBII达电泳纯,分子量为97kD。     

有机胺对阳离子交换树脂性能的影响

采用浸泡方法分别研究了氨、乙醇胺、吗啉对阳离子交换树脂性能的影响。利用光学显微镜观察浸泡前后阳离子交换树脂的形貌。55℃下,分别在pH值为9.3的氨、乙醇胺及吗啉的溶液中浸泡不同时间后离子交换树脂的总交换容量、含水率、湿视密度、湿真密度变化均不明显。以3种不同浓度(0.9mol/L、1.4mol/L、1.9mol/L)的氨、乙醇胺及吗啉的溶液分别浸泡离子交换树脂,浸泡后总交换容量、含水率、湿视密度、湿真密度的变化也不大。在恒定pH值与不同浓度碱化溶液中浸泡后,黑琥珀色半透明离子交换树脂的颜色均变浅。实验结果表明有机胺对凝胶型阳离子交换树脂的性能和形貌影响不大。     

离子交换法纯化重组类人胶原蛋白Ⅱ的研究

用阳离子交换树脂CM-52分离纯化类人胶原蛋白Ⅱ（Human Collagen-like Bioprotein Ⅱ，HCBⅡ）。通过实验，分别确定了静态吸附和柱层析吸附的操作条件，即静态吸附：在pH4．0、NaCl0．15mol／L、进料浓度7g／L，处理量17．26ml／g树脂的条件下进行吸附，吸附时间为60min；柱层析：在pH4．0、NaCl0．15mol／L、进料浓度5g／L、流速5ml／min的条件下进行吸附。然后上柱，在pH4．0、NaCl0．30mol／L下进行脱附。实验表明，采用静态吸附的方式吸附HCBⅡ，然后上柱洗脱，HCBⅡ的吸附量可达到48、6mg／g树脂，回收率83．8％，操作时间短，分辨率高，最终纯化的HCBⅡ达电泳纯，分子量为97kD。     

长白山白眉蝮蛇蛇毒精氨酸酯酶1的研究Ⅰ.纯化、分子量测定及纯度鉴定

利用离子交换 (DEAESephadexA - 50 )和凝胶过滤层析 (SephadexG - 75)技术首次从长白山白眉蝮蛇蛇毒 (AgkistrodonblomhoffiiUssurensis)中纯化得到了一种精氨酸酯酶纯品。经SDS -聚丙烯酰胺凝胶电泳(SDS -PAGE)以及基质辅助激光解吸电离飞行时间质谱 (MALDI/TOF/MS)鉴定为单一纯蛋白 ,分子量为2 991 8.5± 1 5Da ,为进一步研究其结构与功能提供了可靠的依据。     

离子交换树脂层析法分离木糖醇结晶母液

研究了001系列离子交换树脂抗衡阳离子型式、树脂交联度、树脂粒径等因素对木糖醇结晶母液分离为糖组分和糖醇组分的影响.筛选出了具有较好分离效果的 001×8(Ca)(0.15mm～0.30mm)树脂.对柱温、柱长径比、进料量、进料浓度、洗脱速度、洗脱液pH等操作因素进行了分离条件优化.在优化条件下,对未糖醇结晶母液进行分离,得到去除糖组分的糖醇组分回收率为92.3%,完成了木糖醇结晶母液的阶段性分离纯化任务.     

PVP/CHI交联共混水凝胶的pH敏感性研究

制备戊二醛交联的PVP/CH I共混水凝胶,红外光谱佐证了凝胶网络的生成。pH敏感性实验表明,凝胶的溶胀率在pH=1.0的介质中最大,在pH=4.0的介质中次之,在中性或pH=9.0的碱性介质中最低,表明凝胶具有明显的pH敏感性。CH I含量、交联剂用量、CH I脱乙酰度均对凝胶的pH敏感性有影响,CH I含量40%、交联剂用量0.3%、CH I脱乙酰度99%的PVP/CH I凝胶pH敏感性较显著。介质pH值由1.0到7.0变化时,凝胶拉伸强度随着溶胀率的降低而增大。     

两种谷胱甘肽亲和层析介质的制备及其对谷胱甘肽转移酶融合蛋白的纯化

以国产交联琼脂糖6FF为基质,分别以环氧氯丙烷(ECH)、1,4-丁二醇二缩水甘油醚(BDGE)为活化剂,偶联谷胱甘肽(GSH)得到两种连接臂长度不同的GSH亲和层析介质,并以两种自制介质对融合蛋白GST-ADAM15进行了纯化。结果表明:GSH-ECH-琼脂糖凝胶和GSH-BDGE-琼脂糖凝胶的配基密度分别达到了30～35μmol/mL和15～18μmol/mL,经两种介质纯化后的GST融合蛋白,纯度均达到95%以上,BDGE活化对目标蛋白的回收率占总蛋白26%,ECH活化为13%。相对而言,由于连接臂长度的不同,BDGE活化的介质纯化效果优于ECH。     

天然植物中生物碱的提取及树脂法在其纯化中的应用

总结了近期提取和分离纯化方法在天然植物中生物碱类有效成分研究中的应用,特别是对于吸附树脂法在生物碱纯化中的应用进行了系统综述,归纳了具有不同结构的大孔吸附树脂、凝胶型离子交换树脂和大孔型离子交换树脂对生物碱纯化效果的影响规律,进而为高效、高选择性纯化生物碱的专用吸附树脂结构设计和应用提供了可借鉴的研究思路.     

革兰氏阴性菌WaaP蛋白的表达与纯化

采用基因重组方法构建来源于大肠杆菌和铜绿假单胞菌的waa P基因的克隆,利用多种感受态细胞表达出带有不同纯化标签的可溶性Waa P蛋白,并利用亲合层析和凝胶过滤层析对可溶性Waa P蛋白进行纯化,用SDS-PAGE进行检测。对比大肠杆菌和铜绿假单胞菌中Waa P的表达和纯化结果,为蛋白结晶选取能够得到大量稳定和高纯度Waa P蛋白的表达纯化方法,并用该方法,使用硒代甲硫氨酸培养基表达出硒代甲硫氨酸标记的Waa P,为蛋白结构解析时相位的确定提供依据。     

革兰氏阴性菌WaaP蛋白的表达与纯化对比研究

采用基因重组方法构建来源于大肠杆菌和铜绿假单胞菌的waa P基因的克隆,利用多种感受态细胞表达出带有不同纯化标签的可溶性Waa P蛋白,并利用亲合层析和凝胶过滤层析对可溶性Waa P蛋白进行纯化,用SDS-PAGE进行检测。对比大肠杆菌和铜绿假单胞菌中Waa P的表达和纯化结果,为蛋白结晶选取能够得到大量稳定和高纯度Waa P蛋白的表达纯化方法,并用该方法,使用硒代甲硫氨酸培养基表达出硒代甲硫氨酸标记的Waa P,为蛋白结构解析时相位的确定提供依据。     

Understanding and mitigating conductivity transitions in weak cation exchange chromatography

Large conductivity fluctuations were observed during a high pH wash step in a weak cation exchange chromatography process. These conductivity transitions resulted in a conductivity drop during pH increase and a conductivity rise during pH decrease. In some cases, the absolute conductivity change was greater than 6 mS/cm which was sufficient to affect target protein retention on the column. Further investigation revealed that wash buffer concentration, resin ligand density, and resin ligand pK have a profound effect on the magnitude of the conductivity transitions and the shape of corresponding pH traces. A potentiometric electrode selective for sodium ions was used to measure effluent counterion concentrations from two preparative resins during high pH washes, and the number of exchangeable counterions was compared to predictions made using ion exchange equilibrium theory. Results from this analysis show that conductivity transitions can be effectively mitigated without compromising process performance by optimizing the trade-off between wash buffer concentration and wash phase duration.     

Refolding human lysozyme produced as an inclusion body by urea concentration and pH gradient ion exchange chromatography

Summary A new dual-gradient ion exchange chromatographic method was developed to improve the refolding yield of human lysozyme produced inEscherichia coli as an inclusion body. The dissolved and stretched polypeptide chain in a concentrated non-ionic denaturant was adsorbed onto an ion exchange column and induced to refold by gradually decreasing the denaturant concentration and increasing pH in the flowing buffer. The dual gradients of denaturant concentration and pH provided a gradual change of the solution environment along the chromatographic column for the protein to refold, resulting in enhanced activity yield and purity. A post-separation was also studied using size-exclusion chromatography to remove protein aggregates and mis-folded proteins after the refolding step.     

Expanded Bed Adsorption Chromatography for Recovery of Phycocyanins from the Microalga Spirulina Platensis

We carried out the purification of C-phycocyanin and allophycocyanin from Spirulina platensis taking advantage of the adsorption properties of the expanded beds. Initially, phycobiliproteins were released from the microalga cells by osmotic shock. Next, phycocyanins were recovered by applying the centrifuged cell suspension directly to the anion exchanger Streamline-DEAE using expanded bed columns, equilibrated with 50 mM sodium phosphate buffer, pH 7.0. After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and phycocyanins rich solution was eluted with a downward flow of 500 mM sodium phosphate buffer, pH 7.0. Finally, we utilized conventional gel filtration and ion exchange chromatography methods for separation and purification of C-phycocyanin and allophycocyanin. The purification steps were monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the purity of recovered phycocyanins was confirmed by absorption and emission spectroscopy. The main advantage of this new method is the high yield achieved in the steps of product extraction and adsorption by expanded bed adsorption, so reducing both processing times and costs.     

Characterization of ion exchange stationary phases via pH transition profiles

New non-destructive method for characterization of ion exchange chromatographic columns based on transient pH formed by a step change in ionic strength of buffer solutions was examined. The method was used to distinguish between cation and anion or weak and strong ion exchange chromatographic supports and to determine the capacity of the chromatographic resins. The general scheme to distinguish between most commonly used types of ion exchange chromatographic columns was proposed. The duration of pH transient was shown to be linearly proportional to the total ionic capacity and was used to estimate protein dynamic binding capacity of the resin. The effects of pH, concentration and temperature on transient pH duration were examined.     

孔石莼质体蓝素的柱色谱纯化及其N-端氨基酸序列的分析测定

通过DEAE-Sepharose Fast Flow阴离子交换柱和Sephadex G-75凝胶过滤柱分离纯化得到了孔石莼(Ulva pertusa)的质体蓝素。其步骤为:将孔石莼样品以0.02 mol/L磷酸盐缓冲液(pH 7.2)进行匀浆,然后离心去除沉淀,将上清液用硫酸铵分级盐析获得饱和度为40%~80%的盐析蛋白;通过DEAE-Sepharose 柱色谱,在含有0~1.0 mol/L NaCl 的0.01 mol/L磷酸盐缓冲液线性梯度洗脱下,盐析蛋白有3个主要的洗脱峰,然后在Sephadex G-75凝胶过滤色谱柱中进一步纯化。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示,该蛋白质被纯化为单一条带。根据蛋白质电泳迁移率,纯化蛋白质的相对分子质量约为10000。该蛋白质不含糖。纯化的蛋白质经电转移至聚偏二氟乙烯（PVDF）膜后,以Edman降解法进行N-端氨基酸序列测定,前20个氨基酸残基序列为AAIVKLGPDDGSLAFVPSKI。通过对相关蛋白质数据库的检索,发现该序列与3种已报道的海藻的质体蓝素具有较高的序列同源性,其同源性分别为85%,85%和90%。据此,认为孔石莼的质体蓝素已获得纯化,其N-端20个氨基酸残基与已报道的海藻质体蓝素的氨基酸残基有较大的同源性,也存在着一定的变异。     

离子交换树脂在分析化学中的应用

简要评述了离子交换树脂在分析化学中的应用。包括离子交换树脂预富集技术从稀溶液中浓缩痕量离子,离子交换树脂相分光光度法测定微量离子,离子色谱在无机分析中的应用,以及离子交换树脂在有机分析与生化分析中的应用。     

稀土离子在多孔阳离子交换剂内的扩散动力学

研究了稀土离子在Amberlyst15、D001、XN1010多孔树脂内的自扩散。结果表明,扩散过程遵循二级分散扩散机制。用粒内扩散方程求算了有效粒内扩散系数e,将e分解为树脂孔道扩散系数p及树脂固相扩散系数s,p与该离子在外部溶液中的自扩散系数相近,而s接近于与实验用的多孔树脂交联度相同的凝胶树脂内的自扩散系数值。     

INTRAPARTICLE DIFFUSION OF RARE EARTHS IN POROUS CATION EXCHANGERS

Experiments for determining cerium isotope ion exchange rates with macroporous resins Amberlyst 15,D001 and XN1010 are discribed.The kinetics of the isotope ion exchange reaction has been examined by a simple theoretical equation of intraparticle effective diffustivity De in a porous ion exchanger.The ion exchange proceedes by diffusion within the macropores and the solid phase of the resin,De of cerium was affected by the concentration of the bulk solution C and was separated into a macropore diffusivity D^-p and a solid phase diffusivity D^-g by the equation.The diffusion coefficients of the exchanging ion are shown to have the values in the macropores comparable with those in the bulk solution and to have the values in the solid phase comparable with those in gel resin with the same crosslinkage as the resins used for the experiments.     

Preparation, Properties and Mechanism of Inhomogeneous Calcium Alginate Ion Cross-linking Gel Microspheres

Inhomogeneous calcium alginate ion cross-linking gel microspheres,a novel ion absorbent,were prepared by dropping a sodium alginate solution to a calcium chloride solutioin via an electronic droplet generator.Calcium alginate microspheres have uniform particle sizes.a smooth surface and a microporous structure.The electrode probe reveals the inhomogeneous distribution of calcium ions with the highest concentration on the surface,and the lowest concentration in the cores of the spheres.As a novel ion adsorbent,calcium alginate gel microspheres have a lower limiting adsorption mass concentration,a higher enrichment capacity and a higher adsorption capacity for Pb^2 than usual ion exchange resins.The highest percentage of the adsorption is 99.79%.The limiting adsorption mass concentration is 0.0426mg/L.The adsorption capacity for Pb^2 is 644mg/g,Calcium alginate gel microspheres have a much faster ion exchange velocity than D418 chelating resin and D113 polyacrylate resin.The moving boundary model was employed to interpret the ion exchange kinetics process,which indicates that the ion exchange process is controlled by intraparticle diffusion of adsorbable ions.So the formation of inhomogeneous gel microspheres reduces the diffusion distance of adsorbable ions within the spheres and enhances the ion exchange velocity.Alginate has a higher selectivity for pb^2 than for Ca^2 and the selectivity coefficient KCa^Pb is 316. As an ion cross-linking gel,calcium alginate inhomogeneous microspheres can effectively adsorb heavy metal Pb^2 at a higher selectivity and a higher adsorption velocity.It is a novel and good ion adsorbent.     

Simultaneous anion and cation exchange chromatography of whey proteins using a customizable mixed matrix membrane

Membrane chromatography can overcome some of the limitations of packed bed column chromatography but preparation of adsorptive membranes usually involves complex and harsh chemical modifications. Mixed matrix membranes (MMMs) require only the physical incorporation of an ion exchange resin into the membrane polymer solution prior to membrane casting. An advantage of MMMs not previously exploited is that resins with differing adsorptive functionalities can be conveniently embedded within a single membrane at any desired ratio. This presents the opportunity to customize an adsorptive membrane to suit the expected protein profile of a raw feed stream e.g. bovine whey or serum. In this work, a novel mixed mode interaction MMM customized to extract all major proteins from bovine whey was synthesized in a single membrane by incorporating 42.5 wt% Lewatit MP500 anionic resin and 7.5 wt% SP Sepharose cationic resin into an ethylene vinyl alcohol base polymer casting solution. The mixed mode MMM developed was able to bind both basic and acidic proteins simultaneously from whey, with binding capacities of 7.16±2.24 mg α-lactalbumin g(-1) membrane, 11.40±0.73 mg lactoferrin (LF)g(-1) membrane, 59.21±9.90 mg β-lactoglobulin g(-1) membrane and 6.79±1.11 mg immunoglobulin Gg(-1) membrane (85 mg total protein g(-1) membrane) during batch fractionation of LF-spiked whey. A 1000 m(2) spiral-wound membrane module (200 L membrane volume, 1m(3) module volume) is predicted to be able to produce approximately 25 kg total whey protein per h.