两步柱色谱法分离纯化重组肿瘤血管生长抑制因子Kringle 5

建立了一种用Ni2+螯合的Chelating Sepharose Fast Flow亲和柱色谱和Sephadex G-75凝胶排阻柱色谱分离纯化重组肿瘤血管生长抑制因子Kringle 5的方法。采用该工艺得到的重组Kringle 5经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析表明其纯度约为98%,且具有抑制鸡胚绒毛尿囊膜新生血管生成的生物活性。     

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。     

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。     

三步柱色谱法从江浙蝮蛇蛇毒中分离纯化类凝血酶

建立了一种用CM Sepharose CL-6B阳离子交换、DEAE Sepharose Fast Flow阴离子交换和Sephadex G-75凝胶排阻三步柱色谱从江浙蝮蛇蛇毒中分离纯化类凝血酶的方法。在实验室小柱分离方案的基础上,对该纯化工艺进行了放大。当上样量达实验室小柱的25倍时,所得类凝血酶的质量指标与实验室小柱基本一致。采用该法所得的蝮蛇类凝血酶经Shim-pack Diol-300高效凝胶排阻柱测得其相对分子质量约为33500,用Shim-pack VP-ODS反相色谱柱检测其纯度约为96%。从江浙粗蛇毒中提取类凝血酶时,类凝血酶的总质量收率约为0.3%,总活性收率约为64%,比活可达2000 U/mg。     

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。     

从猪血中分离纯化高纯度的猪血红蛋白

为了从猪血中分离纯化高纯度的猪血红蛋白,建立了通过超滤、DEAE-Sepharose Fast Flow离子交换色谱和Sephadex G-75凝胶 排阻色谱三步法制备高纯度猪血红蛋白的方法,并通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、高效凝胶排阻色谱和反相高 效液相色谱方法,对纯化后的猪血红蛋白进行了鉴定。经三步分离纯化后,猪血红蛋白的纯度大于99%,含量为1.328 g/L。     

亲水性高聚物凝胶EGPM分离蛋白质的高速空间排除色谱

利用空间排除色谱分离生物高分子,过去都是采用软的亲水性凝胶为柱填料。例如Bio-Gel P(交联聚丙烯酰胺)、Sephadex(交联葡聚糖凝胶)、Sepharose(珠状琼脂糖)、Bio-Gel A(琼脂糖凝胶)等。这些凝胶不能承受较高的柱压,一般只能在0.002-0.02厘米/秒的流速下操作,才能保持凝胶的机械稳定性。这个流速比高效液相色谱所通用的流速约低一个数量级。因此,达到分离平衡的淋洗时间较长,分离周期往往需要几小时。此外,这类软凝胶一般需要较大的进样量方能检出,如对蛋白质的检测就需要毫克级的量。     

钳蝎毒中抗癫痫肽、镇痛肽和抗肿瘤肽的快速同时分离和鉴定

采用Shim-Pack WCX-1型阳离子交换高压色谱柱对中国东亚钳蝎全蝎毒进行了分离，在鉴定了其中的抗癫痫肽、镇痛肽和抗肿瘤肽活性峰的基础上，应用Shim-Pack DIOL-300型凝胶排阻高压色谱柱对它们进行了进一步分离和鉴定，可以得到较纯的3种多肽。在高压色谱所提供的全蝎毒分离信息的基础上，应用与Shim-Pack WCX-1色谱柱具有相同交换基团的、具有较大吸附容量的CM Sepharose CL-6B软胶介质在低压色谱上对全蝎毒进行了分离，并分别对其中的抗癫痫肽、镇痛肽和抗肿瘤肽进行了鉴定。     

转Bt基因植物表达产物Cry1Ab蛋白的制备纯化方法研究

以转Bt基因水稻为试材,研究其表达产物Cry1Ab蛋白的提取、分离及纯化的方法。实验结果表明,DEAE-纤维素填料对Bt蛋白有较好捕获效果。根据生物信息学方法预测了目标蛋白和主要共存蛋白的等电点和疏水性差异。合理地选择了阴离子交换色谱与疏水作用色谱组合方法。提取液经DEAE-Sephadex A-50柱层析及Phenyl-Sepharose Fast Flow疏水层析分离后,目标蛋白得到了显著的纯化。考察了疏水层析中用不同洗脱液洗脱Cry1Ab蛋白对活性回收率和纯度的影响,结果表明：以0．25mol／L KSCN作洗脱液对活性影响最小,HIC一步纯化倍数可达8倍,总纯化倍数达100倍。     

裙带菜中聚甘露糖醛酸的分离纯化与结构分析

以裙带菜(Undaria pinnatifida, wakame)为原料, 经水提醇沉、DEAE-Sepharose Fast Flow、Sephacryl S-300和Sephacryl S-200凝胶柱分离纯化, 得到2个酸性多糖UPPS03和UPPS04. 高效凝胶渗透色谱测试结果表明, 其为均一多糖, 平均分子量分别为3.6×104和1.1×104. 采用糖组成分析、高碘酸氧化及Smith降解、糖醛酸还原、甲基化、红外光谱和核磁共振等方法对该多糖的化学结构进行了表征. 结果表明, 2个多糖均为1,4连接的聚甘露糖醛酸.     

植物脂多糖的离子交换色谱

研究了应用ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ５０分离米糠提取物中植物脂多糖的方法。结果表明，通过改变体系的离子强度，采用静态吸附和柱色谱都能有效分离脂多糖与一般多糖，柱色谱有脂多糖纯度和吸附效率分别为９８．７％和８８．０％，静态吸附则为９０．５％帮７７．９％。     

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.     

阴离子交换和羟基磷灰石色谱从螺旋藻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藻中提取纯化得到藻蓝蛋白和别藻蓝蛋白。通过比较 ＡＮＸ Ｓｅｐｈａｒｏｓｅ ４ Ｆａｓｔ Ｆｌｏｗ（ｈｉｇｈ ｓｕｂ／ｌｏｗ ｓｕｂ）、 ＤＥＡＥ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ和 Ｑ Ｓａｐｈａｒｏｓｅ 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 ＤＥＡＥＳｅｐｈａｒｏｓｅ 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。     

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.     

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.     

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.     

Mechanism and kinetics of protein transport in chromatographic media studied by confocal laser scanning microscopy. Part I. The interplay of sorbent structure and fluid phase conditions

An experimental study on the interplay of sorbent structure and fluid phase conditions (pH) has been carried out examining adsorption and transport of bovine serum albumin (BSA) and a monoclonal antibody (IgG 2a) on SP Sepharose Fast Flow and SP Sepharose XL. SP Sepharose Fast Flow is characterised by a relatively open pore network, while SP Sepharose XL is a composite structure with ligand-carrying dextran chains filling the pore space. Both adsorbents have similar ionic capacity. Protein transport and adsorption profiles were evaluated using confocal laser scanning microscopy. Under all investigated conditions, BSA uptake could be adequately explained by a pore diffusion mechanism. The adsorption profiles obtained for IgG 2a, however, indicated that changes in fluid phase conditions as well as a change in the solid phase structure could result in a more complex uptake mechanism as compared to pore diffusion alone. This mechanism results in a fast transport of proteins into the adsorbent, followed by an overshoot of protein in the center of the sorbent and a setback towards a homogeneous adsorption profile.     

Affinity purification of proteins using expanded beds.

The use of expanded beds of affinity adsorbents for the purification of proteins from feedstocks containing whole or broken cells is described. It is demonstrated that such feedstocks can be applied to the bed without prior removal of particulate material by centrifugation or filtration thus showing considerable potential for this approach in simplifying downstream processing flow-sheets. A stable, expanded bed can be obtained using simple equipment adapted from that used for conventional packed bed adsorption and chromatography processes. Circulation and mixing of the adsorbent particles is minimal and liquid flow through the expanded bed shows characteristics similar to those of plug flow. Frontal analysis performed with the highly selective affinity system involving the adsorption of human polyclonal immunoglobulin G onto Protein A Sepharose Fast Flow indicate that the adsorption performance of the expanded bed is similar to that achieved when the same amount of adsorbent is used in a packed configuration at the same volumetric flow-rate. The adsorption performance of the expanded bed was not diminished when adsorption was carried out in the presence of intact yeast cells. Batch adsorption experiments also indicated that the adsorption characteristics of the affinity system were not greatly altered in the presence of cells in contrast to results from a less selective ion-exchange system. An expanded bed of Cibacron Blue Sepharose Fast Flow was used to purify phosphofructokinase from feedstock of disrupted yeast prepared by high pressure homogenisation without the need for prior removal of particulate material. The potential for the use of expanded beds in large scale purification systems is discussed.     

Fabrication of Hard Dextran DEAE: Adsorption Equilibria of BSA

A hard dextran-DEAE ion exchanger (Hard Dextran DEAE) was developed. It is hard and keeps good properties of dextran-DEAE for protein separation. It is not compressed in a column and can be used in much wider range of flow rate in the column than the commercial hard gel, DEAE Sepharose Fast Flow made of agarose. The saturation capacity of BSA on Hard Dextran DEAE is about 1.7 times of that on DEAE Sepharose Fast Flow at pH 6.9. Equilibrium isotherm for adsorption of BSA depends on pH considerably. When pH 5.5, the equilibrium isotherm is correlated by the Langmuir equation. When pH 5.05, the isotherm is correlated by the Freundlich equation. The higher the concentration of NaCl is, the smaller the amount of BSA adsorbed. When the concentration of NaCl is higher than 100 mol m–3 at pH 6.9 and 50 mol m–3 at pH 4.8, BSA was not adsorbed on the resin. This may suggest that BSA is adsorbed by electrostatic attraction. About 100 mol m–3 NaCl aqueous solution can be used as an eluant of proteins.