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103.
Since many cell functions are regulated by members of the cytokine receptor superfamily, the artificial mimicry of the cytokine receptor system would be attractive for cellular engineering. We previously showed that an antibody/cytokine receptor chimera can transduce a growth signal in response to non-natural ligands, such as fluorescein-conjugated BSA. However, considerable background of cell proliferation was observed without antigen. Therefore, we redesigned chimeric receptor constructs with different combinations of domains containing anti-fluorescein single chain Fv (ScFv), extracellular D1/D2 as well as transmembrane domains of erythropoietin receptor (EpoR), and the intracellular domain of glycoprotein 130 (gp130), to obtain strictly fluorescein-dependent chimeric receptors. When interleukin-3-dependent Ba/F3 cells were transduced with retroviral vectors encoding individual chimeric receptors, the chimeras either with both D1 and D2 domains or without any EpoR extracellular domain attained a strict ligand-dependent ON/OFF regulation. 相似文献
104.
聚乙二醇对PEG化重组人促红细胞生成素热稳定性和生物学活性的影响 总被引:1,自引:0,他引:1
为了研究聚乙二醇对PEG化rhEPO热稳定性和生物学活性的影响,用酶学方法切除rhEPO和PEGrhEPO分子中的N连接糖基,研究酶切产物在37℃时287nm和350nm吸光度随时间的变化;用MTT比色法,网织红细胞法和HCT法比较体内和体外生物学活性的改变。结果显示聚乙二醇修饰能显著减少无N连接糖基的rhEPO分子间的聚集,降低rhEPO体外生物学活性,但增高体内生物学活性;无N连接糖基的PEG化rhEPO具有与rhEPO相当的体内生物学活性。因此聚乙二醇能提高rhEPO热稳定性;聚乙二醇可替代糖基,维持rhEPO的体内生物学活性。用PEG修饰原核细胞表达的rhEPO是开发rhEPO制品的新思路。 相似文献
105.
Eun Gyo Lee Seung Hui Lee Kyoung Mi Park Jung Eun Baek Jin-Ki Park Won-Kyong Chang Joon-Ki Jung Bong Hyun Chung 《Biotechnology and Bioprocess Engineering》2008,13(2):189-196
The selective removal of impurity proteins and colloidal particles from milk prior to chromatographic purification processes
presents a crucial issue in the production of therapeutic proteins from transgenic animals with high recovery yield and purity.
We have developed an efficient two-step precipitation method for the recovery of the recombinant human erythropoietin (rhEPO)
of interest from transgenic sow milk. Here, rhEPO was partially purified from transgenic sow milk via a two-step precipitation
method consisting of ammonium sulfate and divalent metal precipitations, with a yield of approximately 82.1% and a purification
fold of 10.4 at a copper concentration of 30 mM. Copper proved to be the strongest flocculating agent among the divalent ions
tested for the aggregation of milk proteins under 35%, with ammonium sulfate, zinc, nickel, and calcium demonstrating increasing
flocculating capability in the given order. Copper and zinc proved to be appropriate divalent metals for the recovery of rhEPO
at high yield and purity, and the optimal concentration ranges of copper and zinc were 20~40 and 40~80 mM, respectively. 相似文献
106.
The establishment of erythropoietin (EPO) producing Chinese hamster ovary (CHO) cell lines was conducted using Cre-mediated
cassette exchange. The characterization of site-specific recombination mediated by Cre-recombinase during the cell line development
was also performed. A total of six parental clones, which had various green fluorescence levels ranging from high to low and
containing a single copy of insertion vector (pEGFP-m2), were screened. The EPO targeting vector (pIC-m2-EPO) was targeted into the 6 parental clones by Cre-mediated cassette exchange. Correctly targeted clones were obtained from
4 out of 6 parental clones with 0∼15% of targeting efficiencies. Moreover, there was a positive relationship (R2 = 0.87) between fluorescence levels of the parental clones before Cre-mediated cassette exchange and specific EPO productivities
(q
EPO
) of the correctly targeted clones after Cre-mediated cassette exchange. Therefore, it was verified that the chromosomal loci’s
characteristic gene expression level was not modified even after cassette exchange mediated by Cre recombinase during the
development of EPO producing CHO cell lines. This finding implies that the reproducible development of CHO cell lines largely
producing a desired protein is expected to be achieved by Cre-mediated cassette exchange. 相似文献
107.
红细胞生成素受体研究进展 总被引:2,自引:0,他引:2
红细胞生成素受体是促红细胞生成素的作用受体 ,属于细胞因子超家族的成员。近年来 ,由于其克隆表达技术的应用使人们在对造血机制、EPO的信号转导机制 ,及利用其受体筛选 EPO类似物方面的研究有了新的进展。本文综合近年来有关文献对 EPO受体的研究作一概括性介绍。 相似文献
108.
109.
Enam Abood Sandro Eridani Barbara Sawyer Nigel Westwood Thomas C. Pearson 《Cytotechnology》1991,6(2):131-136
Erythropoietin (Epo) gene expression was studied in a number of different haemopoietic cell lines by in situ hybridization and Northern Blot analysis using a radioisotope-labelled monkey Epo DNA probe. A positive message was expressed by a human cell line, CM-S, derived from a patient with congenital hypoplastic anemia, and by a murine erythro-leukaemic cell line, clone 707, derived from the spleen of Friend virus-infected mice. No message was detected in two megakaryoblastic cell lines, and in a monocytic cell line, derived from a patient with acute monocytic leukaemia. These data may fit with the hypothesis of expression of Epo and other growth factors by haemopoietic cells through a mechanism of so-called autocrine secretion. 相似文献
110.
Ami Oguro Chika KoyamaJing Xu Susumu Imaoka 《Biochemical and biophysical research communications》2014
NADPH-P450 reductase (NPR) was previously found to contribute to the hypoxic response of cells, but the mechanism was not clarified. In this study, we identified a cellular stress response (CSR) as a new factor interacting with NPR by a yeast two-hybrid system. Overexpression of CSR enhanced the induction of erythropoietin and hypoxia response element (HRE) activity under hypoxia in human hepatocarcinoma cell lines (Hep3B), while knockdown of CSR suppressed them. This new finding regarding the interaction of NPR with CSR provides insight into the function of NPR in hypoxic response. 相似文献