4种金属离子亲和层析纯化重组类人胶原蛋白的效果比较

对利用金属离子亲和层析纯化重组类人胶原蛋白过程中使用的金属离子进行了比较,从而对分离纯化的条件进行优化。在相同实验条件下,用4种金属离子柱分离纯化目的蛋白。结果显示,经4种金属离子柱纯化后镍柱的总蛋白收获率最高,铜柱与锌柱居中,钙柱最低;而柱保留时间则为锌柱最高(12.38 m in),钙柱最低(8.25 m in);钙柱洗脱时所需咪唑解离初始浓度最低(100 mmol/L),锌柱最高(200 mmol/L);对SDS-PAGE电泳图进行分析得纯化后类人胶原蛋白的纯度分别为:镍柱82.8%,铜柱83.4%,锌柱96.2%,钙柱94.3%。由此可见锌柱对目标蛋白的亲和力最高,且纯化后类人胶原蛋白的纯度也最高。因此,确定锌离子作为亲和层析纯化重组类人胶原蛋白的金属离子。     

Surface physicochemical characteristics for sodium ion removal with sodium silicate modified magnetite core-shell nanoparticles and activation of their plasmid DNA purification ability

In this study, it is investigated the elimination of residual sodium ions from sodium silicate (Na₂SiO₃) modified Fe₃O₄@SiO₂ nanoparticles, via the additional acidic treatment. Residual sodium ions are generated from the Na₂SiO₃ precursor during the titration for the fabrication of the Fe₃O₄@SiO₂ core-shell structure; however, they were entirely removed after the in situ additional titration at pH 3 using a 1 M of hydrochloric acid (HCl). The thickness of the SiO₂ shell layer of each nanoparticle was measured to 10 nm and the morphology of SiO₂ shell on the Fe₃O₄ core were not significantly different after acid treatment to remove the residual sodium ions. However, the absolute zeta potential of the Na₂SiO₃-modified Fe₃O₄@SiO₂ was decreased about 20% after HCl treatment (?52.5 ± 3.0 mV). In addition, the mean particle size of the Na₂SiO₃-modified Fe₃O₄@SiO₂ was increased (363.98 nm) and the polydispersity index, which indicates dispersibility was increased (0.23) since residual sodium ions were eliminated by the acid treatment. It is assumed that the reducing anions in the diffuse layer of the particle due to the elimination of the residual sodium, as the counter ion, decreased the surface charge, thereby reducing the repulsive force between the Fe₃O₄@SiO₂ nanoparticles. Then Fe₃O₄@SiO₂ nanoparticles were utilized for separating plasmid DNA and separated plasmid DNA was measured with agarose gel electrophoresis and the absorbance monitoring. The efficiency of the DNA purification of the Na₂SiO₃-modified Fe₃O₄@SiO₂ measured to 43.3 ± 5.2 ng/μl which was 58% increased after acid treatment. As a result, the dispersion/magnetic properties and DNA purification efficiency of the Na₂SiO₃-modified Fe₃O₄ became equivalent to the characteristics of the TEOS-modified Fe₃O₄@SiO₂ using the additional acid treatment for sodium ions removal.     

金属亲和膜色谱法纯化木瓜蛋白酶条件优化

建立了一种快速、简便的分离木瓜蛋白酶的方法。尼龙膜经壳聚糖改性后,以金属镍离子N i2+为配基制备了一种新型的蛋白质分离材料,并将该亲和膜应用于木瓜蛋白酶的分离纯化,在对洗脱液、上样速度、洗脱速度、洗脱液的pH值和洗脱液的离子强度进行优化的基础上,成功地分离纯化出木瓜蛋白酶,纯化倍数为20.59倍。     

Comparison of affinity membrane adsorbers for the purification of recombinant human erythropoietin (rhEPO)

In this work affinity membrane adsorbers were investigated for the chromatographic purification of recombinant human erythropoietin (rhEPO) produced in mammalian cells. Cibacron Blue (CB), IDA‐Cu⁺², wheat germ agglutinin (WGA), concanavalin A (ConA) and an anti‐EPO monoclonal antibody (MAb) were tested as affinity ligands, attached to microporous Sartobind^® membranes. In experiments carried out with cell culture supernatant, the best results were obtained with Sartobind–CB, Sartobind–WGA and Sartobind–MAb membranes. The thermodynamic parameters were determined by adsorption isotherms of rhEPO onto the membranes. Sartobind–ConA presented the lowest affinity for rhEPO, as evidenced by a lower association constant. For Sartobind–CB, Sartobind–IDA‐Cu⁺² and Sartobind–MAb K_A was in the order of 10⁵ L mol^?1, whereas for Sartobind–WGA it was 10⁶ L mol^?1. Sartobind–CB eluates were also investigated by RP‐HPLC. The purity level achieved in this one‐step purification strategy was 55%, indicating that the Sartobind–CB membrane is a promising affinity membrane for rhEPO purification. Copyright © 2007 Society of Chemical Industry     

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.     

Preparation of Zn2+‐chelated poly(HEMA‐MAH) cryogel for affinity purification of chicken egg lysozyme

Supermacroporous poly(2‐hydroxyethyl methacrylate) [poly(HEMA)]‐based monolithic cryogel column was prepared by radical cryocopolymerization of HEMA with N‐methacryloyl‐L ‐histidine methyl ester (MAH) as functional comonomer and N,N′‐methylene‐bisacrylamide (MBAAm) as crosslinker directly in a plastic syringe for affinity purification of lysozyme from chicken egg white. The monolithic cryogel containing a continuous polymeric matrix having interconnected pores of 10–50 μm size was loaded with Zn²⁺ ions to form the metal chelate with poly(HEMA‐MAH) cryogel. Poly(HEMA‐MAH) cryogel was characterized by swelling studies, FTIR, scanning electron microscopy, and elemental analysis. The equilibrium swelling degree of the poly(HEMA‐MAH) monolithic cryogel was 5.62 g H₂O/g cryogel. Poly(HEMA‐MAH) cryogel containing 45.8 μmol MAH/g was used in the adsorption/desorption of lysozyme from aqueous solutions. The nonspecific adsorption of lysozyme was very low (7.5 mg/g). The maximum amount of lysozyme adsorption from aqueous solution in phosphate buffer was 209 mg/g at pH 7.0. It was observed that lysozyme could be repeatedly adsorbed and desorbed with the poly(HEMA‐MAH) cyogel without significant loss of adsorption capacity. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

亲合层析法纯化白细胞介素11

白细胞介素 1 1 (IL - 1 1 )是一种具有调节功能的蛋白质。一般采用含有IL - 1 1基因的硫氧还蛋白系统在重组大肠杆菌中来表达制备IL - 1 1融合蛋白 ,产物经金属鏊合层析进行初次分离。本文对融合蛋白IL - 1 1进行亲合纯化工艺研究 ,结果表明 ,IL - 1 1的纯度和回收率均达到较高的水平 ,为后续的纯化奠定了基础     

Optimization of conditions for single-step purification of recombinant hepatitis B surface antigen produced in Pichia pastoris using ion exchange chromatography

ABSTRACT

The adsorption and release of rHBsAg extracted from the final dosage form on various ion exchange resins and under different pH conditions were investigated after its peptide map and isoelectric point (PI) determination. Efficient antigen adsorption to the anion exchange resins occurred when the pH value of the protein buffer was adjusted to 5.0. In purification of rHBsAg derived from the yeast crude extract using Q Sepharose FF column, with adjusting the pH value of the crude extract to 5.0 (i.e., near to the target protein PI) and using 2M NaCl, rHBsAg with high purity (up to >95%) was obtained.

Abbreviations: rHBsAg, recombinant hepatitis B surface antigen; Alhydrogel, aluminum hydroxide; IEF, isoelectric focusing; PI, isoelectric point; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RP-HPLC, reversed-phase high-performance liquid chromatography; SE-HPLC, size-exclusion high-performance liquid chromatography; PBS, phosphate-buffered saline     

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。     

Preparation of an affinity cryogel column for lysozyme purification

Affinity cryogels were synthesized using tris(hydroxymethyl)aminomethane (Tris) as ligand for specific interactions with the lysozyme (LYZ). The cryogel was produced by cryo-copolymerization at ?12°C. A central composite rotational design 2² was used to optimize the immobilization procedure of the Tris on the cryogel. A maximum adsorption for LYZ (149.5 mg/g) was achieved when 376 mg/mL of Tris and 3.06 mol/L of sodium borohydride were used during the Tris immobilization. Chromatographic separation of LYZ from chicken egg white was done with a purity of 92.13%. Results showed that the affinity cryogel was a potential separation medium for LYZ purification.     

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.     

Use of thermolysin metalloprotein affinity metal chromatography in the decontamination of actinide-bearing solutions

Thermolysin metalloprotein affinity metal chromatography (MAMC) has been shown to be effective for the removal and concentration of lanthanide and actinide ions from aqueous solution. Using solution of trivalent lanthanide ions of appropriate radii and of Th⁴⁺ and UO ions as models, the calciumbinding sites of immobilized thermolysin have shown appreciable potential for the decontamination of actinide-bearing waster solutions. The zinc-binding site of the affixed protein may also be used for the removal and concentration of divalent transition metal ions.     

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。     

湿法磷酸脱除金属离子的研究

对氨中和沉淀法脱除湿法磷酸中金属离子进行了实验室和中试研究。优化工艺条件为:氨与P_2O_5质量比15.08%~20.78%,反应温度88~95℃,反应时间0.5~1.0 h,净化磷酸澄清时间≥48 h。中试结果表明,所采用的流程是合理可行的,在一定程度上解决了磷矿品质下降和金属离子升高对磷酸生产及磷肥产品质量的影响。     

Synthesis of monodisperse nonporous crosslinked poly(glycidyl methacrylate) particles with metal affinity ligands for protein adsorption

Monodisperse nonporous crosslinked poly(glycidyl methacrylate) (PGMA) particles with immobilized metal affinity ligands were prepared for selective recovery of proteins. The PGMA particles, with an average size of 2.2 µm, were prepared by a simple dispersion polymerization of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA). The particles were characterized by scanning electron microscopy (SEM) and Fourier‐transform infrared spectroscopy (FTIR). The epoxy groups of the particles were modified with the metal chelating agent iminodiacetic acid (IDA), which forms metal–IDA chelates at the active sites. After charging with copper ions, the particles were used to recover a model protein, bovine hemoglobin (BHb), in a batchwise manner. The particles had the adsorption capacity of 218.7 mg g⁻¹ with little nonspecific adsorption. The adsorption behavior could be described with the Langmuir equation. The effect of pH on the adsorption was also studied. Regeneration of the metal‐chelated particles was easily performed with 50 mmol L⁻¹ ethylenediaminetetraacetic acid (EDTA), followed by washing with water and reloading with Cu²⁺. The particles could be very useful as an affinity separation adsorbent. Copyright © 2005 Society of Chemical Industry     

Preparation of highly magnetic chitosan particles and their use for affinity purification of enzymes

A novel protocol for preparation of highly magnetic chitosan particles, with a coercive force as high as 3500 Oe, has been developed. The surface of the particles was functionalized with aldehyde groups to facilitate the attachment of affinity ligands. The optimum conditions for the preparation of highly magnetic chitosan particles and immobilization of trypsin on magnetic particles were obtained; these particles were then used for affinity purification of aprotinin, and the conditions of affinity purification are discussed in detail. The proposed method was successfully applied to the affinity purification of aprotinin. Copyright © 2003 Society of Chemical Industry     

有机溶剂沉淀法初步提纯谷胱甘肽抽提液的研究

还原型谷胱甘肽(GSH)是生物体内一种具有重要生理功能的活性三肽,其生产多采用发酵法.采用有机溶剂沉淀法对GSH抽提液进行了初步分离提纯.分别采用四氧嘧啶法和高效液相色谱法测定GSH的浓度和纯度,考察了不同有机溶剂和用量、溶液pH、温度和GSH初始质量浓度等对提纯效果的影响,确定了适宜工艺如下:有机溶剂选用乙醇,VR =5,pH的适宜范围为3.0 ～3.4,低温(5℃),较高的初始质量浓度.在上述工艺条件下,GSH纯度可从18.0％提高到41.0％,表明乙醇沉淀法提纯GSH抽提液是有效的,有利于后续分离.     

木器涂料用水性金属离子交联聚氨酯乳液的合成

通过添加ZnO进行交联改性,采用丙酮法合成了聚氯酯乳液。红外光谱分析表明,Zn0已经与体系中的羧基进行了反应,并形成了稳定的结构。凝胶渗透色谱分析表明,ZnO的添加对乳液粒径及其分布影响不大,只有很微小的增加。随着ZnO添加量的增加,所得预聚体的黏度逐渐增加,进而导致分散体的外观变差,但是所得的乳胶膜的硬度逐渐增加。拉伸实验也得出同样的结果,随着ZnO添加量的增加,乳胶膜的机械性能逐渐增强。扫描电镜分析表明,体系中存在着微观相分离。