共查询到20条相似文献,搜索用时 31 毫秒
1.
Zang Y Zhang X Jiang X Li H Zhu J Zhang C Peng W Qin J 《Applied microbiology and biotechnology》2007,74(4):836-842
Thrombopoietin/stem cell factor (TPO/SCF) is a novel fusion protein that combines the complementary biological effects of
TPO and SCF into a single molecule. In this study, TPO/SCF gene was cloned into pET32a and expressed as a thioredoxin (Trx)
fusion protein with a C-terminal 6His-tag in Escherichia coli BL21(DE3) under the control of T7 promoter. Trx-TPO/SCF protein approximately accounted for 20% of the total bacterial proteins
and was found to accumulate in inclusion bodies. Inclusion bodies were separated from cellular debris, washed with buffer
containing 2 M urea, and solubilized with 8 M urea. The refolding of Trx-TPO/SCF was then carried out by an on-column method.
Soluble Trx-TPO/SCF was characterized for its dose-dependent effects on promoting cells proliferation in both TF1 and Mo7e
cell lines. rhTPO/SCF was released by thrombin digestion and further purified by Ni2+ affinity chromatography. Western blot analysis confirmed the identities of Trx-TPO/SCF and rhTPO/SCF. 相似文献
2.
Expression, purification, and characterization of a novel recombinant fusion protein, rhTPO/SCF, in Escherichia coli 总被引:2,自引:0,他引:2
Zang Y Zhang X Yuan D Zhang Y Zhu J Lu H Chang C Qin J 《Protein expression and purification》2006,47(2):427-433
Thrombopoietin (TPO) is the principal regulatory cytokine of megakaryopoiesis and thrombopoiesis and promotes all aspects of megakaryocyte development. Stem cell factor (SCF) is mainly a pleiotropic cytokine acting on hematopoiesis by promoting the survival and proliferation of hematopoietic stem cells and has a potent synergistic effect on megakaryopoiesis in the presence of TPO. Here, we report the construction, expression, and purification of a novel recombinant human thrombopoietin/stem cell factor (rhTPO/SCF) fusion protein, which consists of a truncated human thrombopoietin (1-157 a.a.) plus a truncated human stem cell factor (1-145 a.a.), linked by a peptide (GGGGSPGGSGGGGSGG). The TPO/SCF gene was cloned into the Escherichia coli expression vector pET28a and expressed in BL21(DE3) strain. The rhTPO/SCF constituted up to 6% of the total bacterial protein. Co-expression with E. coli chaperones, Trigger Factor (TF) and GroES/GroEL, and lowering cultivation temperature cooperatively improved the solubility of expressed rhTPO/SCF, resulting in about fourfold increase in the yield soluble rhTPO/SCF. The rhTPO/SCF was purified to homogeneity using anion exchange followed by metal affinity chromatography. Western blot analysis confirmed the identity of the purified protein. rhTPO/SCF stimulated a dose-dependent cell proliferation in both TF1 and Mo7e cell lines. 相似文献
3.
Su L Chen SS Yang KG Liu CZ Zhang YL Liang ZQ 《Protein expression and purification》2006,47(2):477-482
Stem cell factor (SCF) and erythropoietin are essential for normal erythropoiesis and induce proliferation and differentiation synergistically for erythroid progenitor cells. Here, we report our work on construction of SCF/erythropoietin mimetic peptide (EMP) fusion protein gene, in which human SCF cDNA (1-165aa) and EMP sequence (20aa) were connected using a short (GGGGS) or long (GGGGSGGGGGS) linker sequence. The SCF/EMP gene was cloned into the pBV220 vector and expressed in the Escherichia coli DH5alpha strain. The expression level of the fusion protein was about 30% of total cell protein. The resulting inclusion bodies were solubilized with 8 M urea, followed by dilution refolding. The renatured protein was subsequently purified by Q-Sepharose FF column. The final product was >95% pure by SDS-PAGE and the yield of fusion protein was about 40 mg/L of culture. UT-7 cell proliferation and human cord blood cell colony-forming assays showed that the fusion proteins exhibited more potent activity than recombinant human SCF, suggesting a new strategy to enhance biological activities of growth factors. 相似文献
4.
TPO模拟肽与人IgG1 Fc片段的融合表达及其生物学特性研究 总被引:7,自引:0,他引:7
依据本室获得的人TPO模拟肽序列,合成了该模拟肽的DNA序列,分别连接至4种不同长度的人IgG1 Fc基因片段的5′端,并克隆至质粒表达载体pET28a( ),转化大肠杆菌BL21(DE3),筛选获得了4种重组工程菌,其中3种分别高效表达了3种不同长度的融合蛋白,而第4种工程菌未表达,表达的3种融合蛋白的分子量分别约为28kD,12kD和12kD。表达量约占菌体蛋白总量的30%左右,纯化获得了3种TPO模拟肽融合蛋白,3种融合蛋白均有较好的体外活性,维持TPO依赖细胞Ba/F3-mp1生长的EC50分别为:13,10,10nmol/L,用血小板减少症小鼠动物模型,测定了它们的体内活性,3种融合蛋白均有升高血小板和缩短血小板恢复时间的功能,分别比TPO模拟肽活性提高了18,8,8倍,而对白细胞及红细胞无显著影响,分别用3种融合蛋白免疫BALB/c小鼠,均未刺激小鼠产生抗TPO模拟肽抗体,并显示了较好的应用潜力。 相似文献
5.
Stem cell factor (SCF) is a multifunctional cytokine involved in hematopoiesis, melanogenesis and gametogenesis. Previous studies have demonstrated that avian SCF is a requirement for the proliferation and survival of various cell types in vivo and in vitro. In the current study, recombinant quail stem cell factor was produced in Escherichia coli using a prokaryotic expression system. SCF was expressed as a fusion protein with a histidine hexamer tag at the N-terminal end of the protein. Following expression, the protein was purified by affinity chromatography on the Ni-NTA column. The uninduced and induced protein lysates and the purified protein were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Western blot analysis with the monoclonal antibody to the histidine tag identified SCF in the induced cell lysates and the purified sample. The recombinant SCF was approximately 22-23 kD in size. This protein was generated devoid of the signal peptide, the transmembrane domain, and the intracellular domain and, hence, resembles the soluble form of SCF. Biological activity was assayed using the in vitro survival of E12 chicken dorsal root ganglion-derived sensory neurons. The addition of recombinant quail SCF improved neuronal survival. Survival (20.6%) was the highest at the 50 ng/ml concentration of SCF. The availability of quail SCF will be a valuable tool to further resolve the function of stem cell factor in birds. 相似文献
6.
Highly purified proteoglycan (PG) extracted from the nasal cartilage of salmon heads was applied to the ex vivo expansion of hematopoietic progenitor cells prepared from human umbilical cord blood in serum-free cultures supplemented
with the combination of early-acting cytokines, thrombopoietin (TPO), interleukin-3 (IL-3) and stem cell factor (SCF). PG
showed no promoting effects on the cell proliferation rate; however, they promoted the generation of progenitor cells for
granulocyte-macrophages, erythrocytes and/or megakaryocytes in culture with TPO alone or SCF plus TPO. However, no promoting
effect was observed in a combination of IL-3 plus SCF, which showed the highest cell proliferation rate. PG failed to promote
the generation of mixed colony-forming units (i.e. the relatively immature cells in hematopoiesis). These results suggest that PG acts on the relatively mature stem/progenitor
cells, and may function as a regulatory factor in the differentiation pathway of hematopoiesis. 相似文献
7.
重组EpoAB-NGF模拟肽的神经营养功能 总被引:1,自引:0,他引:1
目的:研究重组融合蛋白红细胞生成素AB肽-神经生长因子模拟肽(EpoAB-NGF9和EpoAB-NGF/12)的神经营养作用。方法:构建pET-42a-EpoAB-NGF9/12原核表达质粒,转化大肠杆菌BL21(DE3),以IPTG诱导表达,亲和层析纯化重组蛋白,显微镜观察PC12细胞诱导分化,流式细胞仪检测凋亡细胞。结果:大肠杆菌表达的重组融合蛋白GST-EpoAB-NGF9/12分子量约为30kDa,抗GST抗体免疫印迹反应呈阳性。融合蛋白可以诱导PC12细胞分化,促进轴突生长。R2L1细胞去血清培养,凋亡细胞占(31.7±0.60)%,去血清后加入融合蛋白GST-EpoAB-NGF9/12,细胞凋亡率分别为(25.2±3.52)%、(25.7±1.46)%,呈现一定程度的抑制细胞凋亡的活性。结论:重组EpoAB-NGF模拟肽融合蛋白具有与NGF类似的神经营养作用。 相似文献
8.
Chang Soo Kang Seung-Yeol Son In Seok Bang 《Journal of microbiology (Seoul, Korea)》2008,46(6):656-661
The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine
at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn
(HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector
to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15–20% of the total cell
proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully
by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography,
and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined
by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial
activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the
amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus. 相似文献
9.
Construction of a fusion protein between N-terminal 153 peptide of thrombopoietin and erythropoietin
Thrombopoietin(TPO)isahematopoieticcytokineclonedinrecentyears[1—3].Itsfunctionistoregulatetheformationofplatelet,whichplaysanimportantroleinbloodclotting,bystimulatingthestemcellstodifferentiateanddevelopintomaturemegakaryocytes.Theresultsofanimalexperim… 相似文献
10.
Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo 1p with
its own secretion signal sequence or the α-factor secretion signal sequence, a polyhistidine (6×His) tag for detection, an
enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial
peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy
and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after
proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR. 相似文献
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12.
Frank Sainsbury Philippe Varennes‐Jutras Marie‐Claire Goulet Marc‐André D'Aoust Dominique Michaud 《Plant biotechnology journal》2013,11(9):1058-1068
Studies have reported the usefulness of fusion proteins to bolster recombinant protein yields in plants. Here, we assess the potential of tomato SlCYS8, a Cys protease inhibitor of the cystatin protein superfamily, as a stabilizing fusion partner for human alpha‐1‐antichymotrypsin (α1ACT) targeted to the plant cell secretory pathway. Using the model expression platform Nicotiana benthamiana, we show that the cystatin imparts a strong stabilizing effect when expressed as a translational fusion with α1ACT, allowing impressive accumulation yields of over 2 mg/g of fresh weight tissue for the human serpin, a 25‐fold improvement on the yield of α1ACT expressed alone. Natural and synthetic peptide linkers inserted between SlCYS8 and α1ACT have differential effects on protease inhibitory potency of the two protein partners in vitro. They also have a differential impact on the yield of α1ACT, dependent on the extent to which the hybrid protein may remain intact in the plant cell environment. The stabilizing effect of SlCYS8 does not involve Cys protease inhibition and can be partly reproduced in the cytosol, where peptide linkers are less susceptible to degradation. The effect of SlCYS8 on α1ACT yields could be explained by: (i) an improved translation of the human protein coding sequence; and/or (ii) an overall stabilization of its tertiary structure preventing proteolytic degradation and/or polymerization. These findings suggest the potential of plant cystatins as stabilizing fusion partners for recombinant proteins in plant systems. They also underline the need for an empirical assessment of peptide linker functions in plant cell environments. 相似文献
13.
Natsuko Miura Wataru Aoki Naoki Tokumoto Kouichi Kuroda Mitsuyoshi Ueda 《Applied microbiology and biotechnology》2009,85(2):293-301
We developed a novel method to coat living non-genetically modified (GM) cells with functional recombinant proteins. First,
we prepared GM yeast to secrete constructed proteins that have two domains: a functional domain and a binding domain that
recognizes other cells. Second, we cocultivated GM and non-GM yeasts that share and coutilize the medium containing recombinant
proteins produced by GM yeasts using a filter-membrane-separated cultivation reactor. We confirmed that GM yeast secreted
enhanced green fluorescent protein (EGFP) fusion proteins to culture medium. After cocultivation, EGFP fusion proteins produced
by GM yeast were targeted to non-GM yeast (Saccharomyces cerevisiae BY4741ΔCYC8 strain) cell surface. Yeast cell-surface engineering is a useful method that enables the coating of GM yeast cell surface
with recombinant proteins to produce highly stable and accumulated protein particles. The results of this study suggest that
development of cell-surface engineering from GM organisms (GMOs) to living non-GMOs by our novel cocultivation method is possible. 相似文献
14.
Expression of recombinant proteins on the surface of the coagulase-negative bacterium Staphylococcus xylosus. 总被引:3,自引:0,他引:3
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M Hansson S Sthl T N Nguyen T Bchi A Robert H Binz A Sjlander M Uhln 《Journal of bacteriology》1992,174(13):4239-4245
An expression system to allow targeting of heterologous proteins to the cell surface of Staphylococcus xylosus, a coagulase-negative gram-positive bacterium, is described. The expression of recombinant gene fragments, fused between gene fragments encoding the signal peptide and the cell surface-binding regions of staphylococcal protein A, targets the resulting fusion proteins to the outer bacterial cell surface via the membrane-anchoring region and the highly charged cell wall-spanning region of staphylococcal protein A. The expression system was used to secrete fusion proteins containing sequences from a malaria blood-stage antigen and a streptococcal albumin-binding receptor to the cell surface of S. xylosus. Analysis of the recombinant cells by immunogold staining and immunofluorescence revealed that both the receptor and the malaria peptide were properly processed and exposed on the surface of the host cells. However, only approximately 40 to 50% of the recombinant cells were strongly stained with antiserum reactive with the albumin-binding receptor, while approximately 10 to 15% of the cells were stained with antiserum reactive with the malaria peptide. The incomplete staining of some of the cells suggests steric effects that make the recombinant fusion proteins inaccessible to the reactive antibodies because of variable cell wall structures. However, the results demonstrate for the first time that recombinant techniques can be used to express heterologous receptors and immunogens on the surface of gram-positive cells. 相似文献
15.
Osteopontin (OPN) is a secreted phosphoprotein expressed by many tumor cells, as well as a limited set of normal cells. Native OPN has been shown to support cell adhesion in an RGD-peptide-inhibitable fashion. Here we expressed human OPN in E. coli as a recombinant fusion protein with glutathione-S-transferase (GST). We report that the GST-OPN fusion protein has functional activity. PAP2 (ras-transformed, metastatic murine NIH 3T3) and MDA-MB-435 human mammary carcinoma cells bound to GST-OPN in an in vitro cell adhesion assay nearly as well as to native bovine OPN. Adhesion to the recombinant fusion protein was blocked by addition of GRGDS peptide, suggesting that the cells adhere to the recombinant and native OPN proteins by similar, integrin-mediated mechanisms. Adhesion to both sources of OPN also was inhibited by thrombin treatment of the protein. Thrombin cleaves GST from OPN in the fusion protein, and also cleaves internally in OPN, adjacent to the RGD sequence of the protein. Our results suggest that (a) thrombin cleavage of native OPN may be a natural regulator of OPN function, and (b) the majority of OPN cell binding activity is mediated by the RGD sequence in the protein backbone, with little or no requirement for post-translational modifications that occur in native OPN for adhesive function as measured here. 相似文献
16.
Lee AY Chung HK Bae EK Hwang JS Sung BW Cho CW Kim JK Lee K Han JY Lee CT Youn HJ 《Biotechnology letters》2003,25(3):205-211
Granulocyte-Macrophage colony stimulating factor (GM-CSF) and Granulocyte colony stimulating factor (G-CSF) are cytokines involved in the differentiation of bone marrow progenitor cells into myeloid cells. They also activate mature myeloid cells to mediate a variety of antimicrobial activities and inflammatory responses. Recombinant GM-CSF and G-CSF proteins have been used to treat various diseases including cancer and hematopoietic diseases and to isolate peripheral blood progenitor cells for bone marrow transplantation. A plasmid construct expressing recombinant human G-CSF/GM-CSF fusion protein has now been prepared by linking the human G-CSF and GM-CSF coding regions and the recombinant fusion protein has been successfully expressed in E. coli. The recombinant human G-CSF/GM-CSF fusion protein was extracted and purified from the cellular inclusion and refolded into the biologically active form to show colony stimulating activity. The recombinant fusion protein exhibited colony stimulating activity on human bone marrow cell cultures, indicating that the linkage of GM-CSF and G-CSF by a linker peptide may not interrupt activities of the cytokines in the fusion protein. The colony forming unit of the fusion protein was also higher than those of the cultures treated with the same molar numbers of the recombinant human GM-CSF and G-CSF separately, which suggests that the fusion protein presumably retains both G-CSF and GM-CSF activities. 相似文献
17.
为了探讨血小板生成素(TPO)糖基化与其生物半衰期的关系,首先纯化了巴氏毕赤酵母系统分泌表达的人TPO成熟肽和大肠杆菌表达的人TPON端结构域蛋白.纯化产物经Westernblot鉴定和活性分析后,两种蛋白以相同浓度或相同活性单位给Wister大鼠尾静脉注射,利用酶联免疫分析(ELISA)和TF1细胞测定不同时间点实验动物血浆中TPO浓度或生物学活性,求出两种TPO的生物半衰期(t1/2)值.结果显示,以相同浓度和相同活性单位给药,TPO成熟肽的t1/2均比其N端结构域的长,前者分别是后者的1.65倍和1.27倍. 相似文献
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重组人血小板生成素在大肠杆菌中表达的研究 总被引:2,自引:0,他引:2
采用化学法全合成了编码人血小板生成素(thrombopoietin,TPO)成熟肽N端153氨基酸的基因序列,构建基于该合成基因的表达质粒,结果以谷胱甘肽转硫酶-TPO153(GST-TPO153)融合蛋白的方式获得了占全菌蛋白40%的高效表达.进一步采用PCR方法分别对TPO合成基因及TPOcDNA的翻译起始区(TIR)序列进行定点突变,以降低这一区域的G-C含量.将突变序列分别插入到pBV220表达载体中,重组质粒在转化大肠杆菌JM109后,均获得了表达,其中TIR区突变后的合成基因表达产物约占全菌蛋白的15%.为研究基因下游结构对表达的影响,在不改变氨基酸组成的基础上,构建了TPO合成基因与TPOcDNA的杂合序列表达质粒.研究结果表明翻译起始效率是影响rh-TPO在大肠杆菌中表达的重要因素之一,同时基因下游序列的组成对表达水平也会产生影响. 相似文献