疏水相互作用层析分离重组人干扰素α2b

目的采用疏水相互作用层析分离重组人干扰素α2b,去除干扰素样品中的二聚体,得到高纯度的干扰素用于进一步的研究。方法首先采用阳离子交换层析纯化复性重组人干扰素α2b,去除了大部分的杂蛋白,然后采用疏水相互作用层析纯化重组人干扰素α2b,去除复性过程中产生的错误折叠体和二聚体,并考察盐浓度、pH值、流速和洗脱液中尿素对疏水相互作用层析纯化效果的影响。结果硫酸铵初始浓度1.2 mol/L、缓冲液pH值6.0、流速2.5 mL/min、洗脱液中添加尿素浓度为2 mol/L时疏水相互作用层析纯化效果最佳。最终得到的重组人干扰素α2b非还原型SDS-PAGE电泳均呈单一条带。结论确定了疏水层析纯化重组人干扰素α2b的最优条件,成功提取到具有高活性、高纯度的重组人干扰素α2b纯品。     

疏水作用层析纯化脂肪酶

报道了以对β-硫酸酯己砜基苯胺（SESA）为活化剂制备疏水作用层析剂方法及其柱层析纯化脂肪酶的工艺条件。实验结果表明：活化剂对-β-硫酸酯己砜基苯胺（SESA）最适用量为1．0g／g湿纸纤维素。笨胺：丙酮比为1：4．以0．2mol／L磷酸缓冲液（pH8.0）=1mol／LNaCl为淋洗剂,以0．2mol／L磷酸缓冲液（pH8．0）＋8％吐温80为洗脱刘纯化脂肪酶具有较好的分辨率,酶活回收率64．0％,比活提高6．70倍。     

疏水层析用于大规模纯化重组HBsAg的工艺研究

应用疏水层析法从CHO细胞培养液中纯化HBsAg,每根制备柱每次可处理细胞收液350L,在适宜的上样流速和层析温度条件下,层析后可去除96％的杂蛋白,再经超速离心和凝胶过滤层析,可获HBsAg纯品。经检定,HPLC纯度高于95％,其余各项检定指标均符合《中国生物制品规程》要求。结果表明,此方法纯化效率高、处理样品量大、成本低,适于大规模生产。     

疏水层析纯化人重组白细胞介素－4

应用疏水层析对大肠杆菌表达的人重组白细胞介素－４（ｒｈＩＬ－４）进行了纯化,含有ｒｈＩＬ－４的包涵体,经洗涤、变性、复性后,以Ｂｕｔｙｌ－Ｓｅｐｈａｒｏｓｅ层析,得到了高纯度的ｒｈＩＬ－４．纯度达９７％;回收率为３２％;比活性为２×１０~７Ｕ／ｍｇ,讨论了ｒｈＩＬ－４疏水层析的条件,并对不同的方法纯化白细胞介素－４进行了比较．     

一种新型的琼脂糖疏水层析介质开发及其在重组乙肝表面抗原（CHOHBsAg）分离纯化中的应用

以粒径均一的国产高交联度快速流琼脂糖为基质,采用活化、交联等步骤合成了针对分离纯化CHOHBsAg的3C间臂的丁基琼脂糖疏水介质,通过控制丁基配基密度提高分离HBsAg的纯化倍数和回收率,获得了纯化倍数约20、HBsAg回收率约80%的介质。评估了合成介质的理化性质,流速为500cm/h时柱压力小于0.06MPa,表明介质具有较高的机械强度和良好的流动性能,介质经过酸、碱、变性剂等处理后化学性质稳定。将介质合成工艺进一步放大到2L介质/批,应用到HBsAg分离纯化的三步层析整和工艺中,结果表明,批量合成的疏水介质,HBsAg回收率与进口介质相当, HBsAg终产品纯度在95%以上,符合国家药典要求。最后考察了介质合成批次间的配基密度的可控性和单批次合成介质的重复使用性,结果表明,合成工艺和介质的重复性能满足产业化要求,这种成本低的介质有望替代目前工业生产广泛使用的进口疏水介质。     

一种新型的琼脂糖疏水层析介质开发及其在重组乙肝表面抗原(CHO-HBsAg)分离纯化中的应用

以粒径均一的国产高交联度快速流琼脂糖为基质,采用活化、交联等步骤合成了针对分离纯化CHO-HBsAg的3C间臂的丁基琼脂糖疏水介质,通过控制丁基配基密度提高分离HBsAg的纯化倍数和回收率,获得了纯化倍数约20、HBsAg回收率约80%的介质。评估了合成介质的理化性质,流速为500cm/h时柱压力小于0.06MPa,表明介质具有较高的机械强度和良好的流动性能,介质经过酸、碱、变性剂等处理后化学性质稳定。将介质合成工艺进一步放大到2L介质/批,应用到HBsAg分离纯化的三步层析整和工艺中,结果表明,批量合成的疏水介质,HBsAg回收率与进口介质相当,HBsAg终产品纯度在95%以上,符合国家药典要求。最后考察了介质合成批次间的配基密度的可控性和单批次合成介质的重复使用性,结果表明,合成工艺和介质的重复性能满足产业化要求,这种成本低的介质有望替代目前工业生产广泛使用的进口疏水介质。     

间臂和丁基密度对疏水层析分离纯化重组乙肝病毒表面抗原的影响

以国产高交联度的快流速琼脂糖为基质,合成了针对纯化中国仓鼠卵巢细胞表达乙肝病毒表面抗原(CHO-HBsAg)的不同间臂(3C、8C和10C)和不同配基密度的丁基疏水介质。配基密度的增加和间臂C链的延长都会增加介质的疏水性,从而影响其对CHO-HBsAg的分离效果。采用正交实验考察了配基密度、盐浓度、pH值三个影响因素对不同间臂疏水介质性能的影响。以分离CHO表达的乙肝病毒表面抗原(CHO-HBsAg)的收率和纯化倍数为主要指标。根据正交试验结果,分离效果最佳的介质间臂为8C、配基密度为22μmol/mL。该介质在硫酸铵浓度9%、pH7.0条件下,CHO-HBsAg的收率可以接近100%,纯化倍数达到60。     

活性氧所致超氧化物歧化酶疏水性的变化

用抗坏血酸-Fe(Ⅲ)和过氧化氢分别作用于铜锌超氧化物歧化酶(SOD),经疏水层析分离得到亲水型和疏水型SOD.用胰蛋白酶和胃蛋白酶分别作用于天然SOD,亲水型SOD及疏水型SOD,结果表明疏水型SOD较亲水型SOD及天然SOD易被降解,提示活性氧氧化修饰后的SOD对蛋白水解酶敏感性提高与其疏水性增高有关.     

干扰素-β1b的高效表达、纯化及抗病毒活性研究

IFN-β1b是大肠杆菌产生的17位Cys被Ser替换的人IFN-β的类似物,为了获得高表达,使用了大肠杆菌的偏爱密码子,人工合成了IFN-β-1b基因,插入质粒pBV220中,转化大肠杆菌DH5α.IFN-β1b的制备过程,包括发酵和一系列的纯化步骤.经修饰,IFN-β1b基因在启动子PRPL控制下发酵表达,合成的蛋白质以包涵体的形式存在.培养的细菌经收集、裂解后,将包涵体释放出来,包涵体经含SDS的溶液溶解,DTT还原.纯化过程包括有机溶剂抽提、分子筛层析、脱盐、氧化复性和反相层析,并用旋转蒸发除去有机溶剂.IFN-β-1b在不同种系来源的细胞上显示不同的抗病毒活性.     

A rapid and universal tandem-purification strategy for recombinant proteins

A major goal in the production of therapeutic proteins, subunit vaccines, as well as recombinant proteins needed for structure determination and structural proteomics is their recovery in a pure and functional state using the simplest purification procedures. Here, we report the design and use of a novel tandem (His)(6)-calmodulin (HiCaM) fusion tag that combines two distinct purification strategies, namely, immobilized metal affinity (IMAC) and hydrophobic interaction chromatography (HIC), in a simple two-step procedure. Two model constructs were generated by fusing the HiCaM purification tag to the N terminus of either the enhanced green fluorescent protein (eGFP) or the human tumor suppressor protein p53. These fusion constructs were abundantly expressed in Escherichia coli and rapidly purified from cleared lysates by tandem IMAC/HIC to near homogeneity under native conditions. Cleavage at a thrombin recognition site between the HiCaM-tag and the constructs readily produced untagged, functional versions of eGFP and human p53 that were >97% pure. The HiCaM purification strategy is rapid, makes use of widely available, high-capacity, and inexpensive matrices, and therefore represents an excellent approach for large-scale purification of recombinant proteins as well as small-scale protein array designs.     

十二节杆菌胞外脂肪酶的纯化和性质研究

十二节杆菌发酵得到的胞外脂肪酶,在5L发酵罐经过34h培养,最高酶活可达75 U/mL。通过硫酸铵梯度沉淀和疏水层析纯化,脂肪酶纯化26倍,总得率24.3%。SDS-PAGE显示脂肪酶分子量为33 kD,脂肪酶在40℃和pH 7.0时酶活力最高,同时在24℃经过48h仍保持一半左右的活力。该脂肪酶的酶活受K ,Mg2 激活,而受Zn2 与Co2 的抑制。     

Studies on Some New Thiazolidones as Potential Fungicides

The reaction conditions for the production of d-β-hydroxyphenylglycine (d-HPG) from dl-5-(β-hydroxyphenyl)hydantoin (dl-HPH) by cells of Pseudomonas sp. AJ-11220, and the cultural conditions for this bacterium for the formation of the d-HPG-producing enzyme involved by this bacterium were investigated. The optimal pH of this reaction was about 8.0 and the optimal temperature about 43°C. The d-HPG-producing enzyme was inducibly produced in Pseudomonas sp. AJ-11220 in proportion to the cell growth. Cells containing high activity were obtained when Pseudomonas sp. AJ-11220 was grown in a medium containing 20 g of glucose, 5g of (NH₄)₂SO₄,. 1 g of KH₂PO₄, 3g of K₂HPO₄, 0.5g of MgSO₄–7H₂O, 0.01 g of FeSO₄–7H₂O, 0.01 g of MnSO₄ -4H₂O, 10 g of yeast extract, 5g of dl-5-cyanoethylhydantoin and 20 g of CaCO₃ in a total volume of 1 liter (pH 7.0). Under the optimal conditions, 25 mg/ml of d-HPG was asymmetrically and directly produced from 30 mg/ml of dl-HPH with a molar yield of 92%. Various d-amino acids could also be effectively produced from the corresponding 5-substituted hydantoins.     

用疏水色谱法复性并同时纯化重组人粒细胞-巨噬细胞集落刺激因子

目的：建立一种简单、快速复性并同时纯化大肠杆菌表达的重组人粒细胞一巨噬细胞集落刺激因子（rhGM-CSF）的方法。方法：研究rhGM-CSF在疏水色谱（HIC）上的复性和纯化机理,并对固定相和流动相进行选择和优化,包括固定相配基、流动相中盐的种类、流动相pH值、流动相中还原型谷胱甘肽（GSH）和氧化型谷胱甘肽（GSSG）的比例,以及流动相中尿素的浓度。结果：优化后的固定相为PEG600,流动相中的盐为（NH4）2SO4,流动相pH值为7．0,流动相中添加2．0mol／L尿素、1．8mmol／LGSH和0-3mmol／LGSSG。在优化条件下,HIC可使rhGM-CSF在分离纯化的同时得到复性,比活达1．58×10^7U／mg,纯度为95．7％,质量回收率为56．8％。结论：建立的疏水色谱复性和纯化工艺可简化操作步骤,缩短生产周期。     

Purification of glycogen phosphorylase from small quantities of mouse skeletal muscle

A new approach to the purification of skeletal muscle glycogen phosphorylase is described. The purification scheme is particularly suited to preparation of the enzyme from small amounts of tissue. A combination of dye-ligand chromatography and hydrophobic chromatography yields homogenous enzyme with good recoveries. The purification is rapid and may be completed in a working day.     

Fractionation of Trichoderma Reesei Cellulases by Hydrophobic Interaction Chromatography on Phenyl-Sepharose

Trichoderma reesei cellulase complex was fractionated using hydrophobic interaction chromatography with a phenyl-Sepharose column. Using a linear gradient of ammonium sulphate in the eluent buffer, a selective separation of endoglucanases was obtained at 15 degrees C with a four-fold increase in specific activity.     

Protein instability during HIC: Evidence of unfolding reversibility,and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

A two‐conformation, four‐state model has been proposed to describe protein adsorption and unfolding behavior on hydrophobic interaction chromatography (HIC) resins. In this work, we build upon previous study and application of a four‐state model to the effect of salt concentration on the adsorption and unfolding of the model protein α‐lactalbumin in HIC. Contributions to the apparent adsorption strength of the wild‐type protein from native and unfolded conformations, obtained using a deuterium labeling technique, reveal the free energy change and kinetics of unfolding on the resin, and demonstrate that surface unfolding is reversible. Additionally, variants of α‐lactalbumin in which one of the disulfide bonds is reduced were synthesized to examine the effects of conformational stability on apparent retention. Below the melting temperatures of the wild‐type protein and variants, reduction of a single disulfide bond significantly increases the apparent adsorption strength (～6–8 kJ/mol) due to increased instability of the protein. Finally, the four‐state model is used to accurately predict the apparent adsorption strength of a disulfide bond‐reduced variant. Biotechnol. Bioeng. 2009;102: 1416–1427. © 2008 Wiley Periodicals, Inc.