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Summary A recombinant baculovirus (BmNPV-pk2) was constructed by inserting the human pro-urokinase(pro-UK) cDNA into the genome of baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) adjacent to the strong polyhedrin promoter. The recombinant virus replicated in silkworm larvae, which synthesized 30g pro-UK/ml in the haemolymph within 4 days post-infection. Purification to near homogeneity was accomplished by fractional precipitation with ammonium sulphate and immunoaffinity chromatography with an overall yield of 23% and a specific activity of 100,000IU/mg in fibrin plate assay. This purified product was comprised of a single chain protein with approximately Mr. 50kDa as determined by SDS-PAGE gel. The N-Terminal amino acids sequence revealed that the secretion signal of pro-UK was correctly processed. 相似文献
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影响大肠杆菌中外源基因表达的因素 总被引:41,自引:1,他引:40
大肠杆菌已经被广泛地应用于表达各种外源基因,但是,不同的外源基因在表达效率上却有很大的差异,文章综述了影响大肠杆菌中外源基因表达的因素,这将有助于认识大肠杆菌中外源基因表达的规律,以便采取有效的方法提高外源基因在大肠杆菌中的表达效率. 相似文献
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Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) from a newly characterized thermophile Thermoanaerobacter tengcongensis was expressed in Escherichia coli and purified. Analytical gel filtration suggested that the enzyme exist as a homotetramer in solution. The optimal pH for the forward reaction was found to be 8.0 and the optimal temperature 70 degrees C. The steady-state kinetic characteristics suggest that hypoxanthine is the most effective substrate. This enzyme showed a half-life of 75min at 50 degrees C and no apparent loss of activity after 3 months at 4 degrees C. 相似文献
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Channeling in sulfate activating complexes 总被引:1,自引:0,他引:1
The synthesis of activated sulfate (adenosine 5'-phosphosulfate, APS) and inorganic pyrophosphate from ATP and SO4 is remarkably unfavorable: K(eq) approximately 10(-8) under presumed, near-physiological conditions. Consequently, ATP sulfurylases, which catalyze APS synthesis, suffer approximately 10(8)-fold losses in catalytic efficiency in the forward (APS-synthesis) versus reverse reaction. Losses of this magnitude place this catalyst at risk of being unable to supply its nutrients to the cell in a timely fashion. ATP sulfurylase domains are often embedded in multifunctional complexes that are capable of also catalyzing the second of two steps in the sulfate activation pathway: the phosphorylation of APS to produce PAPS (3'-phosphoadenosine 5'-phosphosulfate). The colocalization of these activities in a single scaffold suggests that evolution might have worked around the inefficiency problem by fashioning a system capable of transferring APS directly between the active sites of the complex, thereby avoiding the solution-phase energetics. For these reasons, representatives from each of the three types of sulfate activating complex (SAC) [Homo sapiens (type I); Mycobacterium tuberculosis (type II); and Rhodobacter sphaeroides (type III)] were tested for the ability to channel APS. A channeling assay that optically detects solution-phase APS was devised with APS reductase from M. tuberculosis, a previously uncharacterized enzyme. Channeling was not detected in two of the three types of SAC; however, the type III SAC channels with high efficiency. Structural models of type III reveal a 75 A-long channel that interconnects active-site pairs in the complex and that opens and closes in response to occupancy of those sites. 相似文献
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组织型纤溶酶原激活剂A链与尿激酶原B链嵌合分子的构建与表达 总被引:1,自引:0,他引:1
利用 D N A 重组技术和定位删除技术,将组织型纤溶酶原激活剂(t P A)的 A 链( Serl Thr263)基因与尿激酶原(pro U K)的 B链( Ser138 Leu411)基因相连,得到嵌合分子基因tu pa,并在昆虫杆状病毒系统中进行表达,表达量可达 500 I U/m l.经单克隆抗体免疫亲和层析纯化细胞表达上清液,得到tu P A 嵌合分子,其比活为 200 000 I U/m g 蛋白. S D S P A G E 及 W estern blot 鉴定证明此表达产物分子量约为 60 k D,与预期值相符.纤维蛋白平板测活及纤维蛋白亲和性分析初步证明,此嵌合分子的溶纤活性与pro U K 相近,而纤维蛋白亲和性高于 pro U K. 相似文献
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噬菌体RB69外切酶活性缺失的DNA 聚合酶突变体(D222A/D327A)在大肠杆菌细胞中表达,表达量达细胞蛋白总量的69% .表达后经DEAE-Sepharose FastFlow , Source 30Q 和HTP三步分离纯化,纯度可达99% 以上.随后测定了该酶利用5种dNTP为底物进行聚合反应的酶促动力学常数(Km 和Kcat),结果表明该酶利用dUTP的能力与利用dTTP的能力相近,Km (dTTP)和Km(dUTP)均较高于其它3种脱氧核苷酸的Km (dATP, dCTP, dGTP),推测其Km 值的差异主要来源于T/U 碱基本身,而并非全部由GC碱基配对与AT碱基配对之间的氢键作用力的强弱差别所决定. 相似文献
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Xiaolan Xu Baocai Xing Meihao Hu Zuoliang Xu Yong Xie Guanghai Dai Jun Gu Yu Wang Zhiqian Zhang 《Cytotherapy》2010,12(2):190-200
Background aimsHepatocellular carcinoma (HCC) recurs with high frequency. Characterization of recurrent HCC cells will facilitate the design of future therapeutic strategies for recurrent HCC.MethodsTwo cell lines, Hep-11 and Hep-12, were established from the same HCC patient's primary and recurrent tumor tissues, respectively, and then analyzed for stem cell-like properties, immune evasion strategies and immunogenicity.ResultsCompared with Hep-11 cells, Hep-12 cells expressed higher levels of liver progenitor cell makers and displayed persistent tumorigenic potential in the serial transplantation assay. Although Hep-12 cells down-regulated human leukocyte antigen (HLA) class I expression, they could still be recognized and killed by autologous-activated tumor-infiltrating lymphocytes (TIL) in vitro. Pre-treatment with cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) increased the expression of HLA class I molecules on Hep-12 cells, and rendered them more susceptible to CD8+ T-cell-mediated recognition and TIL-mediated cytotoxicity in vitro.ConclusionsOur results indicate that Hep-12 cells possess stem cell-like properties, are susceptible to autologous-activated TIL-mediated recognition and cytotoxicity, and pre-treatment with TNF-α and IFN-γ enhances their immunogenicity. This is the first evidence to support the hypothesis that immunotherapy can be used to target recurrent HCC cells with stem cell-like properties. This strategy may be an effective therapeutic approach to prevent HCC recurrence and control recurrent HCC growth. 相似文献
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重组PAI-1在大肠杆菌中的高效表达 总被引:1,自引:0,他引:1
纤溶酶原激活物抑制因子-1(PAI-1)在天然状态下含量很低。为了进行PAI-1的结构与功能的研究,构建了表达重组PAI-1的质粒pBV220/PAI-1,并在大肠杆菌中得到了高效表达。最高表达量为菌体总蛋白量的49%以上,经Western bloting检测,得到了分子量为43.0kDa的反应条带。对形成包涵体的表达产物进行变、复性处理及Sephadex G-75的初步纯化,得到了潜伏态的重组P 相似文献
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将人胰岛素原突变体(A4Glu→Leu)基因重组到pBV220表达载体上,在E.coli系统中得到高效表达,表达产物经SephadexG-50柱层析分离以及胰蛋白酶和羧肽酶B的酶促转化等步骤,可得到纯的人胰岛素突变体(A4Glu→Leu),其氨基酸组成与预期值相符,其受体结合活性及生物活性与标准猪胰岛素的基本相同. 相似文献