阴离子交换和羟基磷灰石色谱从螺旋灌中提纯藻胆蛋白的研究

本文采用离子交换层析和羟基磷灰石吸附层析技术从螺旋灌中提取纯化得到藻蓝蛋白和别藻蓝蛋白。通过比较ANX Sepharose 4Fast Flow(high sub/low sub)、DEAE Sepharose Fast Flow和Q Sepharose Fast Flow等阴离子交换树脂的动态吸附容量以及目标产品的纯度,选用DEAE Sepharose Fast Flow作为层析介质。对离子交换的产物进行了电泳分析,藻蓝蛋白和别藻蓝蛋白的等电点接近,电迁移速率相似。采用羟基磷灰石吸附技术对藻胆蛋白混合物进一步分离纯化,分别得到了藻蓝蛋白和别藻蓝蛋白的纯品,经等电聚焦实验验证显示为均一组成。     

THE 4~Σ- STATES OF THE FO RADICAL

ＴＨＥ４~Σ－ＳＴＡＴＥＳＯＦＴＨＥＦＯＲＡＤＩＣＡＬ￥ＭｉｎｇＢａｏＨＵＡＮＧ（ＤｅｐａｒｔｍｅｎｔｏｆＣｈｅｍｉｓｔｒｙ，ＧｒａｄｕａｔｅＳｃｈｏｏｌ，ＡｃａｄｅｍｉａＳｉｎｉｃａ，Ｂｅｉｊｉｎｇ１０００３９）Ａｂｓｔｒａｃｔ：Ｆｏｒｉｎｖｅｓｔ?..     

Synthesis of New 1,6-disubstituted Fulvenes

ＳｙｎｔｈｅｓｉｓｏｆＮｅｗ１，６－ｄｉｓｕｂｓｔｉｔｕｔｅｄＦｕｌｖｅｎｅｓＨｕＸＩＡＯ；ＳｈｕＦｅｎｇ；ＣＨＥＮａｎｄＸｉａｏＴｉａｎＬＩＡＮＧ（ＩｎｓｔｉｔｕｔｅｏｆＭａｔｅｒｉａＭｅｄｉｃａ，ＣｈｉｎｅｓｅＡｃａｄｅｍｙｏｆＭｅｄｉｃａｌＳｃ...     

SYNTHESIS OF PEG-DENDRIMERIC PEPTIDE

ＳＹＮＴＨＥＳＩＳ　ＯＦ　ＰＥＧ－ＤＥＮＤＲＩＭＥＲＩＣ　ＰＥＰＴＩＤＥＳＹＮＴＨＥＳＩＳＯＦＰＥＧ－ＤＥＮＤＲＩＭＥＲＩＣＰＥＰＴＩＤＥ￥ＤｅＸｉｎＷＡＮＧ；ａｎｄＬｅｉＨＵＡＮＧ（ＩｎｓｔｉｔｕｔｅｏｆＭａｔｅｒｉａＭｅｄｉｃａ，ＣｈｉｎｅｓｅＡ...     

TOTAL SYNTHESIS OF NEOCLAUSENAMIDE AND ITS EPIMER

ＴＯＴＡＬＳＹＮＴＨＥＳＩＳＯＦＮＥＯＣＬＡＵＳＥＮＡＭＩＤＥＡＮＤＩＴＳＥＰＩＭＥＲ￥ＤａｏＦｅｉＨＵＡＮＧ；ＨａｎＳｅｎＬＩＮａｎｄＬｉａｎｇＨＵＡＮＧ（ＩｎｓｈｔｕｔｅｏｆＭａｔｅｒｉａＭｅｄｉｃａ，ＣｈｉｎｅｓｅＡｃａｄｅｍｙｏｆＭｅｄｉｃａ...     

SYNTHESIS OF DIISOFERULIC ACID

ＳＹＮＴＨＥＳＩＳ　ＯＦ　ＤＩＩＳＯＦＥＲＵＬＩＣ　ＡＣＩＤＳＹＮＴＨＥＳＩＳＯＦＤＩＩＳＯＦＥＲＵＬＩＣＡＣＩＤ￥ＣｈｕｎＢｏＡＩａｎｄＬｉａｎＮｉａｎｇＬＩ（ＩｎｓｔｉｔｕｔｅｏｆＭａｔｅｒｉａＭｅｄｉｃａ，ＣｈｉｎｅｓｅＡｃａｄｅｍｙｏｆＭｅｄ...     

STUDY ON SYNTHESIS，CHARACTERIZATION AND CATALYSIS OF PILLARED ANION CLAY（Ⅲ）──SYNTHESIS OF ZnAl－BW_（11）O_（39）Z（H_2）~（n－）

ＳＴＵＤＹＯＮＳＹＮＴＨＥＳＩＳ，ＣＨＡＲＡＣＴＥＲＩＺＡＴＩＯＮＡＮＤＣＡＴＡＬＹＳＩＳＯＦＰＩＬＬＡＲＥＤＡＮＩＯＮＣＬＡＹ（Ⅲ）──ＳＹＮＴＨＥＳＩＳＯＦＺｎＡｌ－ＢＷ_（１１）Ｏ_（３９）Ｚ（Ｈ_２）~（ｎ－）ＴｈａｎｋｓｆｏｒｔｈｅｓｕｐｐｏｒｔｏｆＦｏｕｎｄａｔｉｏｎｏｆＣＥＣａｎｄｔｈｅｏｐｅｎｌａｂｏｆＨｙｄｒｏｔｈｅｒｍａｌＳｙｎｔｈｅｓｉｓｉｎＪｉｎｎＵｎｉｖｅｒｓｉｔｙｅｄｄｉｆｆｅｒｅｎｔｋｉｎｄｓｏｆｔｒｉｈｅｔｅｒｏｐｏｌｙｏｘｏｍｅｔａｌａｔｅ（ＴＨ＇ＰＯＭｓ）ｔｏｔｈｅＺｎＡＩｌａｙｅｒｅｄｃｌａｙｓ，ｕｓｉｎｇｔｈｅｅｓｔｅｒｉｆｉｃａｔｉｏｎｏｆｎ－ｂｕｔｙｌａｌｃｏｈｏｌａｎｄａｃｅｔｉｃａｃｉｄａｓｐｒｏｂｅｒｅａｃｔｉｏｎ．ａｎｄｆｏｕｎｄｔｈａｔｔｈｅｐｉｌｌａｒｅｄｃｏｍｐｏｕｎｄｓｗｅｒｅｍｏｒｅａｃｔｉｖｅａｎｄｓｅｌｅｃｔｉｖｅｔｈａｎｔｈｅｓｏｌｉｄａｃｉｄｃａｔａｌｙｓｔｓｓｕｃｈａｓａｃｉｄｉｃｉｏｎ－ｅｘｃｈａｎｇｅｒｅｓｉｎｓｌ４］．Ｏｎｔｈｅｂａｓｉｓｏｆｐｒｅｖｉｏｕｓｗｏｒｋ．ｗｅｎｏｗｅｎｃａｐｓｕｌａｔｅｔｒｉｈｅｔｅｒｏｐｏｌｙｏｘｏｍｅ     

THE CONVERSION OF SYNGAS ON Fe／MnOⅠ．Efects of Metal Modification on Product Distribution

ＴＨＥＣＯＮＶＥＲＳＩＯＮＯＦＳＹＮＧＡＳＯＮＦｅ／ＭｎＯⅠ．ＥｆｅｃｔｓｏｆＭｅｔａｌＭｏｄｉｆｉｃａｔｉｏｎｏｎＰｒｏｄｕｃｔＤｉｓｔｒｉｂｕｔｉｏｎＧｕａｎＮａｉｊｉａ（ＤｅｐａｒｔｍｅｎｔｏｆＣｈｅｍｉｓｔｒｙ，ＮａｎｋａｉＵｎｉｖｅｒｓｉｔ...     

光固定化藻蓝蛋白对体外肝癌细胞7402的抑制作用

从钝顶螺旋藻中提取、纯化生物活性蛋白－藻蓝蛋白,采用光固定法固定藻蓝蛋白到组织培养聚苯乙烯（ＰＳｔ）基板上,制备生物材料,研究这种新材料对体外肝癌细胞７４０２的抑制作用。实验表明,初始固定化藻蓝蛋白的浓度为２０μｇ／ｅｗｌｌ时,对７４０２细胞的抑制率达到５５％,浓度继续升高,对癌细胞的抑制率反而下降,当藻蓝蛋白浓度高达０．５ｍｇ／ｗｅｌｌ及１ｍｇ／ｗｅｌｌ时抑制率又回升到５５％、６６％,通过显微镜观察到,在固定高浓度蛋白的情况下,组织增减聚苯乙烯表面上的藻蓝蛋白层对肝癌细胞的贴壁生长有一定的阻害作用,可能提高了对癌细胞的抑制率。     

藻胆体核心复合物结构与功能的研究（Ⅱ）

从螺旋藻藻胆体中分离出４种不同种结构和光谱形式的变藻蓝蛋白复合物ＡＰＩ、ＡＰⅡ、ＡＰⅢ和ＡＰＢ，利用吸收光谱、荧光光谱比较了三聚体和单体的光谱特性，通过对吸收光谱的光谱解叠以及各组分的归属，研究了变藻蓝蛋白复合物内各色团间相互作用的性质和在能量传递中的功能，结果表明，复合物内色团间的作用关系可以用Ｆｏｒｓｔｅｒ偶极－偶极作用机制来解释，由于妆蛋白和同源亚基的存在影响其结构的对称性，进而影响各色团间     

二乙氨乙基交联葡聚糖和季氨基琼脂糖色谱分离植物脂多糖的比较

比较了应用Q Sepharose Fast Flow凝胶和DEAE-Sephadex A50凝胶柱色谱分离米糠提取物中米糠脂多糖(LPSR)的效果。结果表明：通过改变体系的离子强度，两凝胶都可使LPSR与一般多糖分离，但仅Q Sepharose Fast Flow柱色谱可实现LPSR与米糠色素的有效分离，并获得淡黄色、99．5％纯度的LPSR产品，而且耐盐性能好于DEAE-Sephadex A50。     

Chemical characterisation of different separation media based on agarose by static time-of-flight secondary ion mass spectrometry

In this paper, the novel application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) for qualitative and semi-quantitative investigation of the surface chemistry of separation media based on beaded agarose is reported. Five different media were studied: DEAE Sepharose Fast Flow, Q Sepharose Fast Flow, SP Sepharose Fast Flow, Phenyl Sepharose Fast Flow at ligand densities between 7 and 33% (w/w) and the base matrix Sepharose 6 Fast Flow. The obtained TOF-SIMS spectra reveal significant chemical information regarding the ligands (DEAE, Q, SP and Phenyl) which are covalently attached to the agarose-based matrix Sepharose 6 Fast Flow. For the anion-exchange media (DEAE and Q Sepharose Fast Flow), the positive TOF-SIMS spectra yielded several strong characteristic fragment peaks from the amine ligands. Structural information was obtained, e.g. from the peak at m/z 173.20, originating from the ion structure [(C2H5)2NCH2CH2NH(C2H5)2l+, which shows that the ligand in DEAE Sepharose Fast Flow is composed of both tertiary and quaternary amines. The positive spectrum of Phenyl Sepharose Fast Flow contained major fragments both from the base matrix and the ligand. The cation-exchanger (SP Sepharose Fast Flow) gave rise to a positive spectrum resembling that of the base matrix (Sepharose 6 Fast Flow) but with a different intensity pattern of the matrix fragments. In addition, peaks with low intensity at m/z 109.94, 125.94 and 139.95 corresponding to Na2SO2+, Na2SO3+ and Na2SO3CH2+, respectively, were observed. The positive TOF-SIMS spectrum of Sepharose 6 Fast Flow contains a large number of fragments in the mass range up to m/z 200 identified as CxHyOz and CxHy structures. The results clearly show that positive TOF-SIMS spectra of different media based on Sepharose 6 Fast Flow are strongly influenced by the ligand coupled to the matrix. The negative TOF-SIMS spectra contained several ligand-specific, characteristic peaks for the cation-exchanger, having sulphonate as the ion-exchange group. Negative fragments such as S-, SO-, SO2-, SO3-, C2H3SO3-, C3H5SO3- and OC3H5SO3- were observed. Phenyl Sepharose Fast Flow, which has an uncharged group (Phenyl) coupled to the agarose matrix yielded one ligand-related peak corresponding to the C6H5O- fragment. DEAE and Q ligands could only be identified by the appearance of the fragments CN- and CNO- in the negative spectrum. However, a strong peak corresponding to the counter ion (Cl-) was observed. TOF-SIMS analysis can also be used for the investigation of residues from the coupling procedure that bonds the ligands to the matrix. One example is the observation of bromine peaks in the negative spectrum of Q Sepharose Fast Flow. Furthermore, it has also been shown that different ligand concentrations of Phenyl Sepharose Fast Flow can easily be detected by TOF-SIMS analysis. Information regarding the difference between the ligand density on the surface of the beads and in the bulk can also be obtained. However, spectra registered on the outermost surface and on the pore surface (crushed beads) of DEAE Sepharose Fast Flow clearly show that the agarose and the DEAE groups are homogeneously distributed in the beads.     

Improved purification of immunoglobulin G from plasma by mixed‐mode chromatography

Efficient loading of immunoglobulin G in mixed‐mode chromatography is often a serious bottleneck in the chromatographic purification of immunoglobulin G. In this work, a mixed‐mode ligand, 4‐(1H‐imidazol‐1‐yl) aniline, was coupled to Sepharose Fast Flow to fabricate AN SepFF adsorbents with ligand densities of 15–64 mmol/L, and the chromatographic performances of these adsorbents were thoroughly investigated to identify a feasible approach to improve immunoglobulin G purification. The results indicate that a critical ligand density exists for immunoglobulin G on the AN SepFF adsorbents. Above the critical ligand density, the adsorbents showed superior selectivity to immunoglobulin G at high salt concentrations, and also exhibited much higher dynamic binding capacities. For immunoglobulin G purification, both the yield and binding capacity increased with adsorbent ligand density along with a decrease in purity. It is difficult to improve the binding capacity, purity, and yield of immunoglobulin G simultaneously in AN SepFF chromatography. By using tandem AN SepFF chromatography, a threefold increase in binding capacity as well as high purity and yield of immunoglobulin G were achieved. Therefore, the tandem chromatography demonstrates that AN SepFF adsorbent is a practical and feasible alternative to MEP HyperCel adsorbents for immunoglobulin G purification.     

基因重组可溶性非融合血管生成抑制剂Kringle 5的色谱分离和纯化

Kringle 5是血纤维蛋白溶酶原中特异抑制内皮细胞增生和迁移活性最高的一种血管生成抑制剂。该实验在前期成功克隆和表达可溶性非融合血管生成抑制剂Kringle 5的基础上,建立了一种两步色谱法分离纯化Kringle 5的方法。首先用SP Sepharose Fast Flow强阳离子交换色谱柱对Kringle 5重组菌体破碎上清液进行初步分离,然后再用丙烯葡聚糖凝胶S-100 HR凝胶排阻色谱柱对其进行进一步的纯化。采用本方法得到的可溶性非融合血管生成抑制剂Kringle 5经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和高效凝胶排阻色谱检测其纯度大于98%,通过鸡胚尿囊膜法确定这种蛋白质具有抑制内皮毛细血管生长的活性。     

Quantitative determination of the ligand content in Benzamidine Sepharose 4 Fast Flow media with ion-pair chromatography

A quantitative hydrochloric acid hydrolysis-HPLC method was developed for the analysis of the ligand content of Benzamidine Sepharose 4 Fast Flow media. The method requires about 100 mg of dried sample and simple reaction vials can be utilised. Release of the ligand (p-aminobenzamidine) from the base matrix (Sepharose 4 Fast Flow) was obtained after hydrolysis for 180min at 70 degrees C in concentrated hydrochloric acid. When Benzamidine Sepharose 4 Fast Flow media were treated this way p-aminobenzoic acid and p-aminobenzamidine were the only products released from the ligand. A chromatographic system based on ion-pair reversed phase separation was used to quantify these ligand products. The mobile phase was made acidic enough to make p-aminobenzoic acid and p-aminobenzamidine positively charged in order to make ion-pair formation with hexanesulfonic acid possible. The relative standard deviation of th e method was below 2% and no systematic errors could be detected when the results were compared to an independent method based on elemental analysis (nitrogen). The new HPLC method was used to analyse ligand densities in the range of 2-20 micromol/ml medium.     

Purification of IgG from Sera of Rabbit and Guinea Pig by Flow-Through Mode Ion-Exchange Chromatography using DEAE Sepharose Fast Flow Column

A one-step purification process using flow-through mode ion-exchange chromatography (Ft-IEC) was evaluated for the purification of Immunoglobulin G (IgG) from rabbit and guinea pig serum. A simple protein precipitation with saturated ammonium sulfate was used to pretreated plasma samples. Chromatographic separation was carried out on DEAE Sepharose Fast Flow (F.F.) column at a flow rate of 1 mL min^?1. Ft-IEC using Tris?CHCl buffer at pH 7.0 allowed the recovery of 22% of the loaded IgG with purity of 95% existed in flowthrough and washing effluents but not in elution effluents. To be compared, bind-elute mode IEC using Tris?CHCl buffer at pH 8.5 based on the routine protocol showed that the recovery of 15% of the loaded IgG with purity of 80% existed in elution effluents. These results indicate that the Ft-IEC is an effective method for purifying IgG from sera of rabbit and guinea pig and may also be suitable for other animal sera by adjusting pH at equilibrium buffer for chromatography column.     

Oscillatory electroosmosis-enhanced intra/inter-particle liquid transport and its primary applications in the preparative electrochromatography of proteins.

The concept of generating an oscillatory electroosmotic flux inside the porous particle to enhance the intra-particle mass transport was presented and a new kind of electrochromatography carried out in a five-compartment electrolyzer were developed. The adsorbent was packed in the central compartment, while the neighboring compartments were used as the elution compartments and the electrode compartments, respectively. Chromatographic separations of human serum albumin on Blue Sepharose Fast Flow, bovine serum albumin (BSA) on DEAE-Sepharose Fast Flow, and BSA on hydroxyapatite were carried out, respectively. The adsorption isotherms were shown to be independent of electric field, while the increase in the electric field strength resulted in a linear increase in the magnitude of electroosmotic flux and the improvement of the breakthrough behavior in all cases. The experiment results have demonstrated the effectiveness of the oscillatory electroosmosis in enhancing intra- and inter-particle mass transport and its high potential to large-scale chromatography.     

两步柱色谱法分离纯化重组猪β2-肾上腺素能受体

β2-肾上腺素能受体是细胞表面受体的一种,它能通过偶联异源三聚体G蛋白将信号转导引入到细胞内部。本实验在成功克隆、表达β2-肾上腺素能受体的基础上,建立了一种两步柱色谱分离纯化目的蛋白质的方法。首先利用Ni2+螯合的高分辨纯化的预带电荷介质Sepharose High Performance与含有六聚组氨酸标签的蛋白质特异结合的性质,对目的蛋白质进行初步分离,接着运用快流速Q琼脂糖凝胶(Quaternary Sepharose Fast Flow)对其进行进一步的分离纯化。采用该方法得到的β2-肾上腺素能受体蛋白质经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和高效凝胶排阻色谱检测其纯度约为95%。结果表明该方法可以对重组猪β2-肾上腺素能受体进行有效的分离纯化。     

Measurement of ligand distribution in individual adsorbent particles using confocal scanning laser microscopy and confocal micro-Raman spectroscopy

Two methods, confocal scanning laser microscopy and confocal micro-Raman spectroscopy were used to analyse the distribution of IgG antibodies immobilized on CNBr-activated agarose beads. In the first method the internal distribution profile of fluorescent labelled Protein A was used as an indirect measure of the distribution of IgG, while the second method detects vibrations originating from aromatic amino acids present in the immobilized antibodies. Both these methods indicate an homogeneous ligand distribution within IgG Sepharose 4 Fast Flow and IgG Sepharose 6 Fast Flow.     

L-asparaginase fromErwinia carotovora

A large-scale process was developed to purify L-asparaginase from submerged cultures of Erwinia carotovora. Cells from 880 L of fermentation broth were harvested and washed using a plate and frame type filter press. A cellular acetone powder was prepared from the washed cells by suspending the cells twice in acetone and the residual acetone was removed by washing the acetone powder in the filter press with 10 mM phosphate buffer (pH 7.0). The cellular acetone powder was extracted with 10 mM borate buffer at pH 9.5. The enzyme-rich borate extract was recovered by filtration and clarified by an in-line bag filter. The filtrate was adjusted to pH 7.5 and filtered through a 1-micron bag filter precoated with Celite and then through a 0.22-micron cartridge filter. The cell-free extract, containing 21 x 10(6) IU of enzyme and 448 g of total protein, was applied to an L-asparagine Sepharose 6 Fast Flow affinity column (9 L) using a bag filter loaded with Cell Debris Remover as an in-line prefilter. The affinity gel was prepared by coupling L-Asn at pH 9.0 to epoxy-activated Sepharose 6 Fast Flow beads. A total of 14 x 10(6) IU of enzyme (35 g protein) was eluted at pH 9.0 in 10.5 L. The eluted enzyme was determined to be greater than 90% pure using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The total process time from whole broth to affinity column elution was 68 h and the enzyme yield was 38%. This improved process for the 880 L fermentation broth produced a cell-free extract of high specific activity, shortened the process time, increased the column capacity, and yielded a product with high purity.