疏水作用色谱法同时纯化及复性基因重组人干扰素-α

 使用高效疏水作用色谱直接从大肠杆菌表达的基因重组人干扰素 α(rhIFN α)包涵体的裂解液中纯化了rhIFN α ,并在纯化的同时获得了高的复性效率 ,使复性和纯化一步完成 ,大大地简化了操作步骤。用凝胶排阻色谱对该法纯化的rhIFN α进行了纯度测定 ,纯度达到 95 %以上。该法的活性回收率分别比稀释法和透析法高 1 0倍和 1 6倍。     

高效疏水相互作用色谱法复性与同时纯化重组人Flt3配体的包涵体蛋白质

Flt3配体(FL)是一类具有促进早期造血功能的细胞因子,在促进造血细胞生长发育及造血动员方面具有重要的临床应用价值。为了用基因工程方法获得大量用于临床和研究的重组人FL(rhFL)蛋白质,本文对在大肠杆菌(E. coli)中表达得到的Flt3配体的包涵体进行回收、洗涤,溶解于8 mol/L脲后在高效疏水相互作用色谱(HPHIC)柱上进行rhFL包涵体的复性与同时纯化,并对其保留特征和复性规律进行了研究。结果表明,在连续进样、变性蛋白质质量浓度为8.51 g/L、固定相选用端基为PEG800、流动相添加4 mol/L脲、1.8 mmol/L 还原型谷胱甘肽(GSH)和0.3 mmol/L氧化型谷胱甘肽(GSSH)、pH 7.0的优化条件下,复性与同时纯化rhFL包涵体的质量回收率为36.9%,纯度达94.5%以上。本文仅用一步HPHIC法成功地复性与同时纯化了rhFL蛋白质,为获得高活性的rhFL产品奠定了一定的工作基础。     

Comparison of the Contributions of Tetrahydrofurfuryl Alcohol and PEG to a-Chymotrypsin Renaturation with High Performance Hydrophobic Interaction Chromatography

Theinvestigationofproteinrenaturation,orrefoldinghasbecomeaveryhotpointinbothliquidchromatography(LC)andlifesciencefields.Besidesusualdilutionanddialysismethods,manynewmethodsconcernproteinrenaturationhavebeenpresented,suchasreversemicelles1,chaperones2,polyethyleneglycol(PEG)3,surfactant4andantibodies5etc.Oneoftheauthorssuggestedhighperformancehydrophobicinter-actionchromatography(HPHIC)tobeanewtoolforproteinrenaturation6.Areviewonproteinrenaturationwithliquidchromatography(LC)wasrecentl…     

Comparison of the Contributions of Tetrahydrofurfuryl Alcohol and PEG to α-Chymotrypsin Renaturation with High Performance Hydrophobic Interaction Chromatography

The contributions of tetrahydrofurfuryl alcohol(THFA) and polyethylene glyco(PEG) to the renature efficiency of α-chymotrypsin were investigated and compared with each other,The maximum increments of bioactivity recovery of α-Chy were found to be 25.1% for THFA,10.4% for PEG,respectively.The experimental results indicated that the denaturant solution containing THFA contributed more to the renaturation of α-Chy in high performance hydrophobic interaction chromatography(HPHIC) than that containing PEG,when the concentration of THFA was 3.2%,the bioactivity recovery of α-Chy is the highest.     

Solubilization and Refolding with Simultaneous Purification of Recombinant Human Stem Cell Factor

Recombinant human stem cell factor (rhSCF) was solubilized and renatured from inclusion bodies expressed in Escherichia coli. The effect of both pH and urea on the solubilization of rhSCF inclusion bodies was investigated; the results indicate that the solubilization of rhSCF inclusion bodies was significantly influenced by the pH of the solution employed, and low concentration of urea can drastically improve the solubilization of rhSCF when solubilized by high pH solution. The solubilized rhSCF can be easily refolded with simultaneous purification by ion exchange chromatography (IEC), with a specific activity of 7.8 × 10⁵ IU·mg⁻¹, a purity of 96.3%, and a mass recovery of 43.0%. The presented experimental results show that rhSCF solubilized by high pH solution containing low concentration of urea is easier to be renatured than that solubilized by high concentration of urea, and the IEC refolding method was more efficient than dilution refolding and dialysis refolding for rhSCF. It may have a great potential for large-scale production of rhSCF.     

高效疏水作用色谱填料的合成及其在重组人干扰素纯化中的应用

高效疏水作用色谱（HPHIC）是利用不同蛋白质表面疏水区域与填料之间具有不同疏水作用进行分离的．由于HPHIC采用盐水体系作为流动相，配体采用极性的有机基团，使蛋白质可以在十分温和的条件下进行分离，且保持其生物活性基本不变[1，2]．自80年代中期以来，HPHIC在蛋白质的分离纯化上得到了广泛的应用．在90年代初期，随着基因工程技术的发展，HPHIC同时也被应用到基因工程的下游纯化技术上[3，4]．本文中我们合成了一OCH2CH3为端基的填料，检验了该填料的分离效果，并利用该填料对酵母菌表达的人αA-干扰素、大肠杆菌（E．col…     

用于变性蛋白质复性及同时纯化的简易型色谱柱的研制及其应用

研制出一种简易型色谱柱并通过装填大颗粒的疏水色谱填料对色谱柱的性能进行了考察,该柱的外形和操作如同传统的液相色谱柱,但它却在生物大分子分离方面有着和高效液相色谱相似的分辨率。另外,只要给该柱装填合适的固定相,比如疏水相互作用色谱固定相,便可用于蛋白质的复性及同时纯化。实验考察了该色谱柱的结构、操作和性能,包括柱压、柱寿命及对蛋白质的分辨率等。以溶菌酶为模型蛋白质,实验测得其在初始浓度为50.0 g/L时的质量回收率和活性回收率分别为(96.6±1.3)%和(101.1±6.0)%。这种简易型色谱柱价格低廉     

重组灵芝免疫调节蛋白的纯化及其性质

采用超滤浓缩、强阴离子交换、疏水作用和凝胶色谱等方法, 对毕赤酵母表达的rGlip进行分离和纯化, 对离子交换色谱中rGlip与固相结合的最佳pH值进行了考察, 并对纯化产物的活性进行了鉴定. rGlip在215 nm处有强的紫外吸收, 经激光解析电离时间飞行质谱鉴定其相对分子量为12722, 经反相液相色谱鉴定纯度≥97%. 设计rGlip的疏水作用色谱, 有效地去除色素. 凝血实验结果表明, rGlip可以凝集绵羊血红细胞, 但对人血A, B, AB和O型等红细胞无凝集作用, 有类似凝集素的生物学活性.     

蛋白折叠液相色谱法复性和纯化大肠杆菌表达的重组人粒细胞集落刺激因子

用蛋白折叠液相色谱法(PFLC)对大肠杆菌表达的包涵体形式的重组人粒细胞集落刺激因子(rhG-CSF)进行了复性并同时纯化。用Cu2+-亚氨基二乙酸（IDA） Sepharose作为固定化金属离子亲和色谱的固定相。在低浓度脲存在下,以咪唑为洗脱剂,采用线性梯度洗脱rhG-CSF。该法仅通过一步PFLC分离,减少了复性过程中rhG-CSF的聚集,复性后的rhG-CSF的比活性为1.8×108 IU/mg,纯度为97%,质量回收率为32%。     

Refolding with Simultaneous Purification of Recombinant Human Granulocyte Colony-stimulating Factor from Escherichia coli Using Strong Anion Exchange Chromatography

Human granulocyte colony-stimulating factor (hG-CSF), a single chain polypeptidecontaining 174 amino acid residues (MW=18,800, pI=6.1), is one of the hemopoieticgrowth factors which plays an important role in stimulating proliferation, differentiation,and functional activation of bloodcells1. It contains a free cysteine at position 17 andtwo intramolecular disulfide bonds, Cys36-Cys42 and Cys64-Cys74, and the two disulfidebonds in G-CSF molecule are both required for its bioactivity2. Wh…