共查询到20条相似文献,搜索用时 15 毫秒
1.
目的:建立rhHPPCn在大肠杆菌中的表达体系,纯化表达产物并检测其体外生物活性。方法:以pUC18-HPPCn为模板,构建原核表达载体pET-24a(+)-HPPCn,转化到大肠杆菌BL21中并诱导表达目标蛋白,离子交换色谱法纯化目标蛋白,Western blot分析其免疫原性,MTT法和3H-TdR掺入法检测其体外活性。结果:成功构建出原核表达载体pET-24a(+)-HPPCn,并诱导使目标蛋白的表达量较高,表达产物中HPPCn 23%以可溶形式存在,两步离子交换后可得纯度为94.2%,Western blot鉴定为目的蛋白,MTT法和3H-TdR掺入法检测其具有明显的促肝癌细胞系增殖活性。结论:成功构建出原核表达载体并实现了目标蛋白高表达,两步离子交换法纯化出所需纯度的蛋白,并验证了其较强的体外促肝癌细胞系增殖活性。 相似文献
2.
Adachi M Higuchi H Miura S Azuma T Inokuchi S Saito H Kato S Ishii H 《American journal of physiology. Gastrointestinal and liver physiology》2004,287(3):G695-G705
Acute ethanol exposure induces oxidative stress and apoptosis in primary rat hepatocytes. Previous data indicate that the mitochondrial permeability transition (MPT) is essential for ethanol-induced apoptosis. However, the mechanism by which ethanol induces the MPT remains unclear. In this study, we investigated the role of Bax, a proapoptotic Bcl-2 family protein, in acute ethanol-induced hepatocyte apoptosis. We found that Bax translocates from the cytosol to mitochondria before mitochondrial cytochrome c release. Bax translocation was oxidative stress dependent. Mitochondrial Bax formed a protein complex with the mitochondrial voltage-dependent anion channel (VDAC). Prevention of Bax-VDAC interactions by a microinjection of anti-VDAC antibody effectively prevented hepatocyte apoptosis by ethanol. In conclusion, these data suggest that Bax translocation from the cytosol to mitochondria leads to the subsequent formation of a Bax-VDAC complex that plays a crucial role in acute ethanol-induced hepatocyte apoptosis. 相似文献
3.
Interleukin-2 (IL-2) is a vital cytokine secreted by activated T lymphocytes, and plays an important role in the regulation
of cellular and immunity of animals. In this study, a gene encoding duck IL-2 was cloned and the soluble recombinant duck
IL-2 (rDuIL-2) was expressed in Escherichia coli via fusion with glutathione S-transferase (GST). The results indicated that the GST-rDuIL-2 fusion protein expressed in E. coli Origami (DE3) was confirmed to be of about 40 kDa molecular mass by SDS-PAGE and western blotting. In order to produce soluble
rDuIL-2 in a low-cost, nontoxic and high-level expression process, lactose was used as a substitute for Isopropyl-β-D-thiogalactopyranoside
(IPTG) to induce the above recombinant strain Origami (pGEX-DuIL-2). By optimizing the expression conditions, the production
of soluble GST-rDuIL-2 fusion protein was about 29% of total cellular soluble proteins, which was similar with IPTG used as
inducer. The soluble GST-rDuIL-2 fusion protein was purified by one-step affinity chromatography, and GST was removed by thrombin.
Then rDuIL-2 was purified by a second affinity chromatography. As a result, the 95% pure rduIL-2 was obtained, and the yield
of rDuIL-2 was about 10.6 mg/l bacterial culture. The bioactivity of rduIL-2 was determined by lymphocyte proliferation assay
in vitro. Our study provided a feasible and convenient approach to produce soluble and biologically active rDuIL-2, which
would be used as an immunoadjuvants for enhancing vaccine efficacy. 相似文献
4.
New procedures for purification of L-asparaginase with high yield from Escherichia coli 总被引:9,自引:0,他引:9
l-Asparaginase is now known to be a potent antineoplastic agent in animals and has given complete remission in some human leukemias. Extensive clinical trials of this enzyme, however, were not possible in the past because of inadequate production of this substance. We have developed practical procedures for producing l-asparaginase in yields of sufficient quantity and purity for more extensive clinical evaluation. The nutritional requirements for optimal production of biologically active l-asparaginase by a strain of Escherichia coli have been ascertained. The highest yields of enzyme were obtained when cells were grown aerobically in a corn steep medium. Good enzyme production was associated with media containing l-glutamic acid, l-methionine, and lactic acid. The addition of glucose to the medium, however, resulted in depressed production of l-asparaginase. Sodium ion appeared to suppress l-asparaginase production. With the procedure described for isolation of biologically active l-asparaginase from E. coli, stable l-asparaginase preparations with a specific activity of 620 IU per mg of protein (1,240-fold purification with 40% total recovery) were obtained. 相似文献
5.
6.
Khattar SK Gulati P Kundu PK Singh V Bughani U Bajpai M Saini KS 《Protein and peptide letters》2007,14(8):756-760
The conditions were optimized for maximum soluble yield of biologically active recombinant p38alpha mitogen activated protein kinase (MAPK) vis-à-vis insoluble fraction (inclusion body formation). This study reports a rapid, economical and single step purification process for the overproduction of GST tagged p38alpha MAPK. A yield of 18 mg of highly purified and soluble protein per liter of bacterial culture within 6 h timeframe was achieved. The purified protein was found to be biologically suitable for phosphorylation by upstream kinases and was catalytically active. We further demonstrated that our in-house p38alpha MAPK is more potent (>30%) than a commercially available enzyme. 相似文献
7.
Balduino KN Spencer PJ Malavasi NV Chura-Chambi RM Lemke LS Morganti L 《Molecular biotechnology》2011,48(3):228-234
Aggregation is a serious obstacle for recovery of biologically active heterologous proteins from inclusion bodies (IBs) produced
by recombinant bacteria. E. coli transformed with a vector containing the cDNA for Bothropstoxin-1 (BthTx-1) expressed the recombinant product as IBs. In
order to obtain the native toxin, insoluble and aggregated protein was refolded using high hydrostatic pressure (HHP). IBs
were dissolved and refolded (2 kbar, 16 h), and the effects of protein concentration, as well as changes in ratio and concentration
of oxido-shuffling reagents, guanidine hydrochloride (GdnHCl), and pH in the refolding buffer, were assayed. A 32% yield (7.6 mg
per liter of bacterial culture) in refolding of the native BthTx-1 was obtained using optimal conditions of the refolding
buffer (Tris–HCl buffer, pH 7.5, containing 3 mM of a 2:3 ratio of GSH/GSSG, and 1 M GdnHCl). Scanning electron microscopy
(SEM) showed that that disaggregation of part of IBs particles occurred upon compression and that the morphology of the remaining
IBs, spherical particles, was not substantially altered. Dose-dependent cytotoxic activity of high-pressure refolded BthTx-1
was shown in C2C12 muscle cells. 相似文献
8.
Jian Feng Li Jie Zhang Zhen Zhang Hong Wei Ma Jia Xin Zhang Shuang Quan Zhang 《The protein journal》2010,29(5):314-319
The human beta defensins-4 (hBD4) exhibit a broad range of antimicrobial properties and are thought to be ideal therapeutic
agents because of their potential ability to circumvent the problems of acquired resistance often observed with other antimicrobial
therapies. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and
purification of cationic antibacterial peptide hBD4. The fusion protein expressed in a soluble form was purified to a purity
of 90% by Ni-IDA chromatography and 637 mg protein of interest was obtained per liter of fermentation culture. After the SUMO-hBD4
fusion protein was cleaved by the SUMO protease at 30 °C for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally,
about 166 mg recombinant hBD4 was obtained from 1 L fermentation culture with no less than 96% purity and the recombinant
hBD4 had similar antimicrobial properties to the synthetic hBD4. Thus, the SUMO-mediated peptide expression and purification
system potentially could be employed for the production of recombinant cytotoxic peptides. 相似文献
9.
Barbara Manconi Tiziana Cabras Alberto Vitali Chiara Fanali Antonella Fiorita Rosanna Inzitari Massimo Castagnola Irene Messana Maria Teresa Sanna 《Protein expression and purification》2010,69(2):219-225
This work reports the successful recombinant expression of human statherin in Escherichia coli, its purification and in vitro phosphorylation. Human statherin is a 43-residue peptide, secreted by parotid and submandibular glands and phosphorylated on serine 2 and 3. The codon-optimized statherin gene was synthesized and cloned into commercial pTYB11 plasmid to allow expression of statherin as a fusion protein with intein containing a chitin-binding domain. The plasmid was transformed into E. coli strains and cultured in Luria–Bertani medium, which gave productivity of soluble statherin fusion protein of up to 47 mg per liter of cell culture, while 112 mg of fusion protein were in the form of inclusion bodies. No significant refolded target protein was obtained from inclusion bodies. The amount of r-h-statherin purified by RP–HPLC corresponded to 0.6 mg per liter of cell culture. Attenuated total reflection-Fourier transform infrared spectroscopy experiments performed on human statherin isolated from saliva and r-h-statherin assessed the correct folding of the recombinant peptide. Recombinant statherin was transformed into the diphosphorylated biologically active form by in vitro phosphorylation using the Golgi-enriched fraction of pig parotid gland containing the Golgi-casein kinase. 相似文献
10.
Qi Wang Cui Min Tingting Yan Hefang Pu Yinqiang Xin Shuangquan Zhang Lan Luo Zhimin Yin 《World journal of microbiology & biotechnology》2011,27(11):2603-2610
l-glutamine (Gln) is an important conditionally necessary amino acid in human body and potential demand in food or medicine
industry is expected. High efficiency of l-Gln production by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system
has been developed. We report here first the application of small ubiquitin-related modifier (SUMO) fusion technology to the
expression and purification of recombinant Bacillus subtilis GS. In order to obtain GS with high Gln-forming activity, safety and low cost for food and pharmaceutics industry, 0.1% (w/v)
lactose was selected as inducer. The fusion protein was expressed in totally soluble form in E. coli, and expression was verified by SDS–PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel
nitrilo-triacetic acid (Ni–NTA) resin chromatography with a yield of 625 mg per liter fermentation culture. After the SUMO/GS
fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 121 mg
recombinant GS was obtained from 1 l fermentation culture with no less than 96% purity. The recombinant purified GS showed
great transferase activity (23 U/mg), with 25 U recombinant GS in a 50 ml reaction system, a biosynthesis yield of 27.5 g/l l-Gln was detected by high pressure liquid chromatography (HPLC) or thin-layer chromatography. Thus, the application of SUMO
technology to the expression and purification of GS potentially could be employed for the industrial production of l-Gln. 相似文献
11.
A novel production method in Escherichia coli for an antimicrobial peptide of 21 amino acids, buforin IIb, which is a synthetic analog of buforin II, has been developed.
The buforin IIb gene was cloned into the vector pET32a to construct an expression vector pET32a–buforin IIb. The fusion protein
Trx-buforin IIb, purified by nickel nitrilo-triacetic acid (Ni-NTA) resin chromatography, was cleaved by hydroxylamine hydrochloride
to release recombinant buforin IIb. Purification of recombinant buforin IIb was achieved by HPLC: about 3.1 mg/l active recombinant
buforin IIb with purity >99% was obtained. The recombinant buforin IIb showed antimicrobial activities that were similar to
the synthetic one. 相似文献
12.
Xiaoping Wu Haishan Tian Yadong Huang Sixian Wu Xiaoju Liu Cong Wang Xiaojie Wang Zhifeng Huang Jian Xiao Wenke Feng Xiaokun Li 《Applied microbiology and biotechnology》2009,82(3):439-444
A rapid and efficient expression and purification system has been developed for large-scale production of biologically active
recombinant human keratinocyte growth factor-2 (rhKGF-2). The gene encoding human KGF-2 was cloned into the expression vector
pET3c and transformed into Escherichia coli BL21(DE3)/pLys S. Under optimal conditions in a 30-l fermentor, the average bacterial yield and the average expression level
of rhKGF-2 of three batches were up to 732 g and 32%, respectively. The recombinant protein was purified by cation exchange
and heparin-affinity chromatography. One hundred and sixty five milligrams of pure rhKGF-2 was achieved per liter culture.
A preliminary biochemical characterization of purified rhKGF-2 was performed by Western blotting and mitogenic activity analysis,
and the results demonstrated that purified rhKGF-2 could react with anti-human KGF-2 antibody and stimulate the proliferation
of HaCat cells.
Xiaoping Wu and Haishan Tian contributed equally to this work. 相似文献
13.
Abdalla A. Elshereef Andr Jochums Antonina Lavrentieva Lena Stuckenberg Thomas Scheper Drte Solle 《Engineering in Life Science》2019,19(11):730-740
High cell densities for transient transfection with polyethyleneimine (PEI) can be used for rapid and maximal production of recombinant proteins. High cell densities can be obtained by different cultivation systems, such as batch or perfusion systems. Herein, densities up to 18 million cells/mL were obtained by centrifugation for transfection evaluation. PEI transfection efficiency was easily determined by transfected enhanced green fluorescence protein (EGFP) reporter plasmid DNA (pDNA). A linear correlation between fluorescence intensity and transfection efficiency was improved. The transfection efficiency of PEI was highly dependent on the transfection conditions and directly related to the level of recombinant protein. Several factors were required to optimize the transient transfection process; these factors included the media type (which is compatible with low or high cell density transfection), the preculture CHO‐K1 suspension cell density, and the pDNA to PEI level. Based on design of experiment (DoE) analyses, the optimal transfection conditions for 10 × 106 cells/mL in the CHOMACS CD medium achieved 73% transfection efficiency and a cell viability of over 80%. These results were confirmed for the production of transforming growth factor‐beta 1 (TGF‐β1) in a shake flask. The purified TGF‐β1 protein concentration from 60 mL supernatant was 27 µg/mL, and the protein was biologically active. 相似文献
14.
Tsun-Hsien Hsiao Chia-Jen Lin Yi-Shao Chung Gang-Hui Lee Tseng-Ting Kao Wen-Ni Chang Bing-Hung Chen Jan-Jong Hung Tzu-Fun Fu 《Molecular and cellular biology》2014,34(3):498-509
Alcoholism induces folate deficiency and increases the risk for embryonic anomalies. However, the interplay between ethanol exposure and embryonic folate status remains unclear. To investigate how ethanol exposure affects embryonic folate status and one-carbon homeostasis, we incubated zebrafish embryos in ethanol and analyzed embryonic folate content and folate enzyme expression. Exposure to 2% ethanol did not change embryonic total folate content but increased the tetrahydrofolate level approximately 1.5-fold. The expression of 10-formyltetrahydrofolate dehydrogenase (FDH), a potential intracellular tetrahydrofolate reservoir, was increased in both mRNA and protein levels. Overexpressing recombinant FDH in embryos alleviated the ethanol-induced oxidative stress in ethanol-exposed embryos. Further characterization of the zebrafish fdh promoter revealed that the −124/+40 promoter fragment was the minimal region required for transactivational activity. The results of site-directed mutagenesis and binding analysis revealed that Sp1 is involved in the basal level of expression of fdh but not in ethanol-induced upregulation of fdh. On the other hand, CEBPα was the protein that mediated the ethanol-induced upregulation of fdh, with an approximately 40-fold increase of fdh promoter activity when overexpressed in vitro. We concluded that upregulation of fdh involving CEBPα helps relieve embryonic oxidative stress induced by ethanol exposure. 相似文献
15.
Sakamoto S Putalun W Pongkitwitoon B Juengwatanatrakul T Shoyama Y Tanaka H Morimoto S 《Plant cell reports》2012,31(1):103-110
A single-chain variable fragment antibody (scFv) against plumbagin (PL) accumulated the PL production in the hairy roots of
Plumbago zeylanica. Recombinant Agrobacterium rhizogenes (ATCC 15834) containing an scFv gene against PL (PL-scFv) were obtained through triparental mating and transformed into P. zeylanica to induce PL-scFv protein in the hairy roots. Up to 40 μg recombinant PL-scFv were expressed per milligram of soluble protein
in transgenic P. zeylanica hairy root cultures. The mean PL content obtained from transgenic hairy roots (12.24 μg/100 mg dry weight) exhibited 2.2
times higher than those obtained from wild-type (5.48 μg/100 mg dry weight). The high correlation between the PL-scFv expression
level and PL content of the recombinant plants suggested that the PL biosynthesis pathway had been modulated by the expression
of PL-scFv protein in the hairy roots of P. zeylanica. 相似文献
16.
Production of bioactive human hemangiopoietin in <Emphasis Type="Italic">Escherichia coli</Emphasis>
To devise an efficient approach for production of human hemangiopoietin (hHAPO), the gene of hHAPO was synthesized and subcloned
into the pSUMO vector with a SUMO tag at the N-terminus. The expression construct was then transformed into the expression
strain E. coli BL21(DE3). The fusion protein was expressed in soluble form and identified by SDS-PAGE and Western blotting. The fusion protein
was purified to 90% purity by metal chelate chromatography with a yield of 45 mg per liter fermentation culture. The SUMO
tag was removed by cleavage with SUMO protease at room temperature for 1 h, and the hHAPO was then re-purified by the metal
chelate chromatography. Finally, about 21 mg hHAPO was obtained from 1 liter of fermentation culture with no less than 95%
purity. The recombinant hHAPO significantly stimulated the proliferation of human umbilical vein endothelial cells. 相似文献
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19.
Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and
angiogenesis. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin)
in Escherichia coli by expressing via fusion with solubility-promoting peptides and optimizing the expression conditions. The rhEndostatin was expressed via fusion with glutathione S-transferase (GST) and NusA protein, respectively. It revealed that NusA protein enhanced the production
of soluble rhEndostatin; but GST didn’t. By optimizing the expression conditions, the production of soluble NusA-rhEndostatin
fusion protein was about 50% of total cellular proteins and about 90% of the products appeared in the cellular supernatant
fraction. The soluble NusA-rhEndostatin fusion protein was purified by one-step hydrophobic interaction chromatography and
NusA was removed by thrombin. Then rhEndostatin was purified by affinity chromatography and gel filtration chromatography.
As a result, a simple and economical purification procedure for rhEndostatin isolation was obtained. The biological activity
of the rhEndostatin was demonstrated in vitro using a human vascular endothelial cells (HuVECs) proliferation assay. Our study provides a feasible and convenient approach
to produce soluble and biologically active rhEndostatin. 相似文献
20.
Angiogensis can be blocked by inhibitors such as endostatin and angiostatin. The kringle 5 fragment of plasminogen also has a potent inhibitory effect on endothelial cell proliferation and leads to the inhibition of angiogenesis. It has promise in anti-angiogenic therapy due to its small size and potent inhibitory effect. Preparation of kringle 5 has been achieved through the proteolysis of native plasminogen and recombinant DNA technology. Bacterially expressed recombinant kringle 5 is mainly insoluble and expressed at low level. The refolding yield is also low. To produce recombinant human kringle 5 in a large quantity, we have genetically modified a strain of Pichia pastoris. On methanol induction, this strain expressed and secreted biologically active, recombinant kringle 5. The expression level of the engineered strain in culture reached more than 300mgl-1. Purification was easily achieved by precipitation, hydrophobic and DEAE ion exchange chromatography. The recovery of recombinant kringle 5 was about 50% after purification. Yeast-expressed kringle 5 has a higher activity in anti-endothelial proliferation than bacterially expressed kringle 5.Revisions requested 9 November 2004; Revisions received 2 December 2004 相似文献