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浙江大学学报（医学版） 2009年第3期38卷 260-264页

作者：沈其陈枢青浙江大学药学院药理毒理与生化药学研究所浙江杭州310058

目的:纯化HSA/IL1ra融合蛋白并对其生物学活性进行检测。方法:采用亲和层析及离子交换层析分离纯化HSA/IL1ra融合蛋白,检测其对IL1引起的对A375 S2的杀伤保护作用。结果:获得的HSA/IL1ra融合蛋白HPLC纯度在98%以上,并且具有与天然IL1ra... 详细信息

目的:纯化HSA/IL1ra融合蛋白并对其生物学活性进行检测。方法:采用亲和层析及离子交换层析分离纯化HSA/IL1ra融合蛋白,检测其对IL1引起的对A375 S2的杀伤保护作用。结果:获得的HSA/IL1ra融合蛋白HPLC纯度在98%以上,并且具有与天然IL1ra相似的、对IL1杀伤A375 S2细胞的保护作用。结论:获得高纯度的HSA/IL1ra融合蛋白并且具有相应的生物学活性。

关键词：受体,白细胞介素1/拮抗剂和抑制剂血清白蛋白重组融合蛋白质类/分离和提纯重组融合蛋白质类/生物合成黑色素瘤

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重组人Tum-5融合蛋白的克隆、表达、纯化及鉴定

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第四军医大学学报 2005年第14期26卷 1279-1281页

作者：马楠张英起颜真第四军医大学药学系生物技术中心陕西西安710033

目的:构建人肿瘤抑素(tumstatin)活性片段Tum5的融合重组表达载体并对其进行诱导表达和蛋白纯化以获得大量重组融合蛋白.方法:人工合成Tum5基因,进行测序分析,将该基因克隆入原核表达载体pQE30中,转化感受态细胞DH5α,经IPTG诱导表达重... 详细信息

目的:构建人肿瘤抑素(tumstatin)活性片段Tum5的融合重组表达载体并对其进行诱导表达和蛋白纯化以获得大量重组融合蛋白.方法:人工合成Tum5基因,进行测序分析,将该基因克隆入原核表达载体pQE30中,转化感受态细胞DH5α,经IPTG诱导表达重组融合蛋白,对表达产物进行TricineSDSPAGE电泳分析和WesternBlot检测分析.结果构建了重组融合表达质粒pQE30Tum5,表达的蛋白经TricineSDSPAGE电泳分析,在约Mr10×103处出现了一条新生的蛋白条带,经灰度扫描检测,表达量约占菌体总蛋白的40%,纯化后的蛋白灰度扫描检测,纯度为95%.结论:成功地构建了人Tum5基因的原核表达载体,并纯化了该基因的原核表达产物.

关键词： Tumstatin Tum-5 重组融合蛋白质类/分离和提纯 pQE载体

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重组人C22orf37融合蛋白的克隆、表达、纯化及鉴定

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第四军医大学学报 2009年第12期30卷 1057-1059页

作者：叶星明姜昌丽张英起第四军医大学药学系生物技术中心陕西西安710033

目的:构建人C22orf37基因的原核表达载体,表达并纯化C22orf37重组蛋白.方法:应用PCR方法从人外周血白细胞cDNA文库中获得人C22orf37基因,成功构建人C22orf37融合表达载体,诱导表达His-C22orf37融合表达蛋白并对表达产物进行SDS-PAGE电... 详细信息

目的:构建人C22orf37基因的原核表达载体,表达并纯化C22orf37重组蛋白.方法:应用PCR方法从人外周血白细胞cDNA文库中获得人C22orf37基因,成功构建人C22orf37融合表达载体,诱导表达His-C22orf37融合表达蛋白并对表达产物进行SDS-PAGE电泳分析和Western Blot鉴定.应用镍螯合层析法纯化重组融合蛋白,并对纯化产物进行纯度分析.结果:成功构建了人C22orf37重组融合载体pQE30-C22orf37,工程菌pQE30-C22orf37/DH5α经IPTG诱导后的菌体蛋白经SDS-PAGE电泳分析,在Mr约18 000处出现了一条新生的蛋白条带,Western Blot结果显示该条带能够与His抗体特异性结合.应用Ni-NTA层纯化出目的蛋白,纯化后的蛋白经Primer Premier软件分析纯度约80%.结论:成功构建了人C22orf37的融合蛋白原核表达载体并成功表达与纯化了人C22orf37的融合蛋白,为下一步的C22orf37 mAb的制备和C22orf37蛋白表达的组织分布及功能研究奠定了基础.

关键词：人C22orf37基因原核表达重组融合蛋白质类/分离和提纯

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重组葡激酶-水蛭素融合蛋白的分离纯化工艺建立

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安徽医科大学学报 2010年第4期45卷 567-569页

作者：张光满秦宜德于爱平安徽医科大学生物化学与分子生物学教研室合肥230032 军事医学科学院放射与辐射医学研究所北京100850

目的建立大肠杆菌高密度表达的重组葡激酶-水蛭素融合蛋白(STH)分离纯化工艺。方法高密度培养重组大肠杆菌并诱导表达STH,离心收集菌体,反复冻融后收集上清液经超滤、两步阴离子交换层析,分离纯化STH目的蛋白。结果纯化后的STH纯度为98... 详细信息

目的建立大肠杆菌高密度表达的重组葡激酶-水蛭素融合蛋白(STH)分离纯化工艺。方法高密度培养重组大肠杆菌并诱导表达STH,离心收集菌体,反复冻融后收集上清液经超滤、两步阴离子交换层析,分离纯化STH目的蛋白。结果纯化后的STH纯度为98%以上,纤溶比活性2.6×104IU/mg,活性回收率56%。结论建立了STH的纯化工艺,该纯化工艺简便,时程短,重复性好,适合于大规模生产。

关键词：重组融合蛋白质类/分离和提纯色谱法,离子交换

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Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第14期275卷 9963-9969页

作者： Yusof, R Clum, S Wetzel, M Murthy, HMK Padmanabhan, R Univ Kansas Med Ctr Dept Biochem & Mol Biol Kansas City KS 66160 USA Univ Alabama Birmingham Ctr Macromol Crystallog Birmingham AL 35294 USA

Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia cole as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni2+ affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gin residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.

关键词：氨基酸序列氨基酸类二氨基/代谢色谱法亲和色谱法凝胶克隆分子辅酶类/代谢保守序列登革热病毒/酶学内肽酶类/代谢酶激活大肠杆菌动力学蛋白质前体/分离和提纯蛋白质前体/代谢 RNA解螺旋酶类重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢丝氨酸内肽酶类/代谢底物特异性病毒非结构蛋白质类/化学病毒非结构蛋白质类/分离和提纯病毒非结构蛋白质类/代谢

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Baculovirus-transfer vector for eukaryotic expression and immuno-affinity purification of Gal4-fusion proteins

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BIOTECHNIQUES 1999年第5期26卷 808-+页

作者： Chen, GQ Courey, AJ Univ Calif Los Angeles Dept Chem & Biochem Los Angeles CA 90095 USA

No abstract available

关键词：氨基酸序列杆状病毒科/遗传学碱基序列碱性螺旋-环-螺旋转录因子类生物技术细胞系克隆分子 DNA引物/遗传学 DNA 重组/遗传学 DNA结合蛋白质类/遗传学真菌蛋白质类/遗传学真菌蛋白质类/分离和提纯基因表达遗传载体免疫印迹法重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯阻遏蛋白质类/遗传学限制性内切酶图谱法酿酒酵母蛋白质类斜纹夜蛾属转录因子/遗传学转录因子/分离和提纯动物

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Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand

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BIOTECHNOLOGY PROGRESS 1999年第2期15卷 283-286页

作者： Cha, HJ Dalal, NG Pham, MQ Bentley, WE Univ Maryland Dept Chem Engn College Pk MD 20742 USA Univ Maryland Inst Biotechnol Agr Biotechnol Ctr College Pk MD 20742 USA

The fusion protein of green fluorescent protein (GFP) and human interleukin-2 (hIL-2) was produced in insect Trichoplusia ni larvae infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was composed of a metal ion binding site (His)(6) for rapid one-step purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv), enterokinase cleavage site for recovering hIL-2 from purified fusion protein, and hIL-8 protein. The additional histidine residues on fusion protein enabled the efficient purification of fusion protein based on immobilized metal affinity chromatography. In addition to advantages of GFP as a fusion marker, GFP was able to be used as a selectable purification marker;we easily determined the correct purified fusion protein sample fraction by simply detecting GFP fluorescence.

关键词：生物学标记/分析肠肽酶/代谢基因表达绿色荧光蛋白质类指示剂和试剂/代谢昆虫/化学昆虫/生长和发育白细胞介素2/遗传学白细胞介素2/分离和提纯白细胞介素2/代谢幼虫发光蛋白质类/遗传学发光蛋白质类/分离和提纯发光蛋白质类/代谢金属/代谢重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢动物人类

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Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase α in vitro

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第48期274卷 34493-34498页

作者： Isotani, S Hara, K Tokunaga, C Inoue, H Avruch, J Yonezawa, K Kobe Univ Biosignal Res Ctr Nada Ku Kobe Hyogo 6518501 Japan Massachusetts Gen Hosp Diabet Unit Boston MA 02129 USA Massachusetts Gen Hosp Med Serv Boston MA 02129 USA Harvard Univ Sch Med Boston MA 02129 USA

p70 S6 kinase alpha (p70 alpha) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70 alpha activation in a manner that can be overcome by coexpression of p70 alpha with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70 alpha polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70 alpha phosphorylation in vitro is accompanied by a substantial restoration in p70 alpha kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70 alpha by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70 alpha activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined.

关键词：细胞系培养基无血清/药理学酶激活/药物作用免疫印迹法有丝分裂原/药理学突变磷酰化/药物作用磷酸转移酶类(醇族体)/遗传学磷酸转移酶类(醇族体)/分离和提纯磷酸转移酶类(醇族体)/代谢沉淀素试验蛋白激酶类蛋白质丝氨酸苏氨酸激酶/遗传学蛋白质丝氨酸苏氨酸激酶/代谢重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢核糖体蛋白质S6激酶类/遗传学核糖体蛋白质S6激酶类/代谢 TOR丝氨酸-苏氨酸激酶人类

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A highly active, soluble mutant of the membrane-associated (S)-mandelate dehydrogenase from Pseudomonas putida

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BIOCHEMISTRY 1999年第38期38卷 12367-12376页

作者： Xu, Y Mitra, B Wayne State Univ Sch Med Dept Biochem & Mol Biol Detroit MI 48201 USA

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a member of the flavin mononucleotide-dependent alpha-hydroxy acid oxidase/dehydrogenase family, is a membrane-associated protein, in contrast to the more well-characterized members of this protein family including glycolate oxidase (GOX) from spinach. In a previous study [Mitra, B., et al. (1993) Biochemistry 32, 12959-12967], the membrane association of MDH was correlated to a 53 amino acid segment in the interior of the primary sequence by construction of a chimeric enzyme, MDH-GOX1, in which the membrane-binding segment in MDH was deleted and replaced with the corresponding 34 amino acid segment from the soluble GOX. Though MDH-GOX1 was soluble, it was an inefficient, nonspecific enzyme that involved a different transition state for the catalyzed reaction from that of the wild-type MDH. In the present study, it is shown that the membrane-binding segment in MDH is somewhat shorter, similar to 39 residues long. Partial or total deletion of this segment disrupts membrane localization of MDH. This segment is not important for substrate oxidation activity. A new chimera, MDH-GOX2, was created by replacing this shorter membrane-binding segment from MDH with the corresponding 20 amino acid segment from GOX. The soluble MDH-GOX2 is very similar to the wild-type membrane-bound enzyme in its spectroscopic properties, substrate specificity, catalytic activity, kinetic mechanism, and lack of reactivity toward oxygen. Therefore, it should prove to be a highly useful model for structural studies of MDH.

关键词：醇氧化还原酶类/拮抗剂和抑制剂醇氧化还原酶类/遗传学醇氧化还原酶类/分离和提纯醇氧化还原酶类/代谢氨基酸序列细胞膜/酶学酶激活/药物作用酶激活/遗传学大肠杆菌/酶学大肠杆菌/遗传学黄素单核苷酸/化学黄素单核苷酸/代谢羟丁酸盐类/化学动力学膜蛋白质类/遗传学膜蛋白质类/分离和提纯膜蛋白质类/代谢分子序列数据肽碎片/遗传学肽碎片/分离和提纯肽碎片/代谢蛋白质结合/遗传学假单胞菌恶臭/酶学假单胞菌恶臭/遗传学重组融合蛋白质类/化学合成重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯溶解度立体异构现象

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Poly(ADP-ribose) polymerase is a B-MYB coactivator

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第14期275卷 10692-10696页

作者： Cervellera, MN Sala, A Consorzio Mario Negri Sud Dept Mol Pharmacol & Pathol I-66030 Santa Maria Imbaro Chieti Italy

B-MYB is implicated in cell growth control, differentiation, and cancer and belongs to the MYB family of nuclear transcription factors. Evidence exists that cellular proteins bind directly to B-MYB, and it has been hypothesized that B-MYB transcriptional activity may be modulated by specific cofactors. In an attempt to isolate proteins that interact with the B-MYB DNA-binding domain, a modular domain that has the potential to mediate protein-protein interaction, we performed pull-down experiments with a glutathione S-transferase-B-MYB protein and mammalian protein extracts. We isolated a 110-kDa protein associated endogenously with B-MYB in the nuclei of HL60 cells. Microsequence analysis and immunoprecipitation experiments determined that the bound protein was poly(ADP-ribose) polymerase (PARP). Transient transfection assays showed that PARP enhanced B-MYB transactivation and that PARP enzymatic activity is not required for B-MYB-dependent transactivation. These results suggest that PARP, as a transcriptional cofactor of a potentially oncogenic protein, may play a role in growth control and cancer.

关键词：结合部位细胞周期蛋白质类细胞核/代谢 DNA结合蛋白质类/化学 DNA结合蛋白质类/分离和提纯 DNA结合蛋白质类/代谢谷胱甘肽转移酶 HL-60细胞哺乳动物癌基因蛋白质类/代谢骨肉瘤聚ADP核糖聚合酶类/遗传学聚ADP核糖聚合酶类/分离和提纯聚ADP核糖聚合酶类/代谢蛋白质生物合成重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢反式激活因子类/化学反式激活因子类/分离和提纯反式激活因子类/代谢转录遗传肿瘤细胞培养的动物人类

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