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1.
S-腺苷甲硫氨酸合成酶的组成型表达、产物纯化及鉴定   总被引:2,自引:0,他引:2  
将大肠杆菌(E.coli K12) S 腺苷甲硫氨酸合成酶(SAMS)基因克隆至质粒pBR322中,获得的重组质粒pBR322-SAMS转入大肠杆菌JM109菌株,构建了能高效组成型表达SAMS的重组菌E.coli JM109 (pBR322-SAMS)。将重组大肠杆菌破碎后上清液经20%~40%硫酸铵分级盐析、Phenyl-Sepharose Fast Flow疏水层析和Q Sepharose Fast Flow离子交换层析,即可得到纯度提高5倍,比活为48.7 μ/mg的SAMS,三步纯化的总回收率为62%,纯度达到92%。SAMS表达量为1 176μ/L,占到菌体可溶性总蛋白的20%。重组酶的最适反应pH为8.5,4℃下在pH 7.5的缓冲液中保温10h酶活性几乎不改变。重组酶反应的最适温度为55℃ ,酶活性稳定的温度范围为20~35℃。重组酶的KmL Met为0.22mmol/L,Vmax L-Met为1.07mmol/(L·h),Km ATP为0.52 mmol/L,Vmax ATP为1.05 mmol/( L·h)。  相似文献   

2.
人血红素加氧酶-1(human heme oxygenase-1,hHO-1)是血红素代谢的限速酶,直接调节体内胆红素水平。利用软件Spdbv程序对hHO-1进行结构模拟预测,以Ala取代第25位His残基,hHO-1活性部位的结构有较明显的改变,但据模拟推测突变后hHO-1与血红素仍然具有结合性。依据模拟结果,构建野生型和突变体表达载体pBHO-1和pBHO-1(M),并分别转化大肠杆菌DH5α,IPTG诱导表达目的蛋白。表达产物经30%-60%(NH4)2SO4盐析后纯度提高3.6倍,再经两次Q-Sepharose Fast Flow阴离子交换树脂分离则表达产物的纯度提高30倍。酶活性测定显示,突变体hHO-1(△hHO-1)较野生型hHO-1(whHO-1)活性下降了91.21%。本研究显示hHO-1的第25位His在酶与底物血红素氧化反应中起着重要作用,为有效调控酶活性发挥其生物学作用提供依据。  相似文献   

3.
Coagulation factor VIII (FVIII) concentrates are used in the treatment of patients with Hemophilia A. Human FVIII was purified directly from plasma using anion exchange chromatography followed by gel filtration. Three Q-Sepharose resins were tested, resulting in 40% recovery of FVIII activity using Q-Sepharose XL resin, about 80% using Q-Sepharose Fast Flow and 70% using the Q-Sepharose Big Beads. The vitamin K-dependent coagulation factors co-eluted with FVIII from the anion exchange columns. In the second step of purification, when Sepharose 6FF was used, 70% of FVIII activity was recovered free from vitamin K-dependent factors.  相似文献   

4.
人肿瘤抑素(Tumstatin)在E.coli中的克隆、表达及活性分析   总被引:1,自引:0,他引:1  
从人胚肾2 93细胞中扩增肿瘤抑素(tumstatin)基因,进行原核表达,纯化和生物活性检测.利用原核表达载体pMAL c2在大肠杆菌BL2 1中表达肿瘤抑素,经AmyloseResin亲和层析柱和QSepharoseFastFlow柱纯化,通过体外内皮细胞增殖、内皮细胞凋亡和鸡尿囊绒膜新生血管生成试验检测其抑制活性.MBP tumstatin在BL2 1中表达率约2 0 % ,肿瘤抑素纯度可达95 % .肿瘤抑素可明显抑制内皮细胞增殖(IC50 约为15 μg ml)、诱导内皮细胞凋亡和抑制鸡尿囊绒膜新生血管生成.研究结果表明,肿瘤抑素对内皮细胞具有明显的抑制作用,提示其在肿瘤治疗中有潜在的应用前景.  相似文献   

5.
Measurement of adsorption breakthrough curves in packed beds has shown that the amounts and rates of uptake of immunoglobulin M (IgM) onto the commonly used anionic ion-exchanger Q Sepharose Fast Flow(based on 6% agarose) are severely limited as a result of the large molecular size of this adsorbate(RMM 950000). A similar ion-exchanger based on a more porous 4% agarose, Q Sepharose 4 Fast Flow was evaluated as an alternative adsorbent for the purification of IgM. Equilibrium adsorption isotherms and the effective diffusivities of IgM within these two adsorbents were measured. Q-Sepharose 4 Fast Flow was found to have a maximum capacity for IgM 2.5 times greater than that of Q Sepharose 6 Fast Flow and the effective diffusivity of IgM was found to be between 6 and 7 times greater than with the latter material. Comparison of the breakthrough curves obtained for these adsorbents at a variety of flow velocities confirm that Q Sepharose 4 Fast Flow is a superior adsorbent for the capture and purification of large proteins.This revised version was published online in October 2005 with corrections to the Cover Date.  相似文献   

6.
Human paraoxonase (hPON3) is a high density lipoprotein-related glycoprotein with multi-enzymatic properties and antioxidant activity which is proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. In this study, hPON3 gene was amplified from Human Fetal Liver Marathon-Ready cDNA and expressed in Escherichia coli. A majority of the expressed protein existed as inclusion bodies. The inclusion bodies were solubilized with Triton X-100 and refolded in vitro. The refolded rhPON3 was purified by DEAE-Sepharose Fast Flow and its purity was up to 90%. The Km and Vmax values of refolded rhPON3, in respect to phenylacetate hydrolysis were 7.47 +/- 2.14 mM and 66 +/- 17 U/min/mg (n = 3). The Km and Vmax values of refolded rhPON3, in respect to dihydrocoumarin hydrolysis were 0.83 +/- 0.21 mM and 621 +/- 66 U/min/mg (n = 3). The refolded rhPON3 exhibited similar antioxidant activity to that of rhPON3 purified from the soluble fraction of cell lysate and could effectively protect LDL from Cu2+ induced oxidation.  相似文献   

7.
An intracellular protease from a bacterium, Bacillus pumilus HL721, was purified about 5000-fold by Chromatography with a Q-Sepharose Fast Flow column, TSK-gel HA-1000 glass column, and TSK-gel G3000SWXL column using Bz-Gly-Ala-Pro as a substrate. The enzyme was the most active at pH around 7.5 and stable from 4.5 and 8.0. The enzyme activity was inhibited by Cu2+, EDTA, N-ethylmaleimide, o-phenanthroline, and p-chloromercuribenzoic acid. The molecular weight of the enzyme was 155,000 by gel filtration. The enzyme removed dipeptide from the carboxyl end of some peptides used as substrates. From these results the enzyme seems to be a dipeptidyl carboxypeptidase.  相似文献   

8.
In this work, we present the production of an active 43 aa recombinant human beta-defensin-1 (rhBD-1(43)) in Escherichia coli AD202 cells using specific pLMM1-rhBD-1 expression system. Unique solubility properties of the C-terminal fragment of light meromyosin (LMM) allowed us to overcome foreseeable problems with isolation procedures and toxicity caused by rhBD-1 to the host organism. As a result, the majority of fusion protein (LMM-rhBD-1(43)) was obtained in the soluble state, isolated by a low salt-high salt treatment of total cell protein. The rhBD-1(43) was cleaved from the fusion with Protease 4 and purified on CM Sepharose Fast Flow column with the yield of approximately 1 mg rhBD-1(43) from 6 g of wet weight cells. Purified rhBD-1(43) showed antimicrobial activity against E. coli ML-35p at a concentration of 129 microM. The procedure of rhBD-1 expression and purification we present can provide a reliable and simple method for production of different cationic peptides for biological studies.  相似文献   

9.
Human brain serine racemase (hSR) was expressed in large amounts in E. coli with N-terminal His-tag (His-hSR). His-hSR expressed in inclusion body was solubilized and purified to homogeneity by Ni-NTA affinity column. Purified His-hSR was refolded in Tween 20/cycloamylose with approximately 50% efficiency, and refolded His-hSR was isolated by Q Sepharose column chromatography. The refolding conditions are described in detail. His-hSR catalyzed the elimination of L-Ser as well as L-Ser-O-sulfate to form pyruvate.  相似文献   

10.
 Neurturin (NTN)是新近发现的一种神经营养因子 ,是 GDNF家族的成员之一 .将 5′端引入了羟胺切割位点的 h NTN基因克隆到硫氧还蛋白融合表达载体 p Thio His A,在宿主菌 BL2 1中获得了稳定、高效表达 ,表达产物以包涵体形式存在 .在变性条件下经羟胺切割、柱层析纯化后复性 ,获得纯度达 90 %以上的 rh NTN.经鸡胚背根神经节 (DRG)培养法测定具有生物学活性 .  相似文献   

11.
The lymphokine human interleukin 4 (IL-4) has been expressed from a plasmid in the cytoplasm of Escherichia coli. Advantage has been taken of insolubility of the human IL-4 in E. coli for rapid purification of this protein in only a few steps. We describe extraction and renaturation procedures which solubilize human IL-4 yielding biologically active protein. The protein was purified to homogeneity by one passage over a gel-filtration column. The refolded human IL-4 was characterized by N-terminal sequence analysis, amino acid analysis and bioassays. The refolded E. coli-derived human IL-4 has biological activity on T and B cells and binds to the human IL-4 receptor, comparable to mammalian expressed human IL-4, indicating that the protein is folded correctly.  相似文献   

12.
The domains of protein G from streptococcus which bind immunoglobulin G have been cloned and expressed in Escherichia coli (Fahnestock et al., 1986). Because protein G binds to several animal immunoglobulin G's, it has many immunochemical applications. This report describes process development for large-scale production of this recombinant protein G (also known as GammaBind G). In 200 l cultures of E. coli, this protein G variant was released from the cell into the culture medium by heating at 80 degrees C for 10 min. The concentration was monitored by either a competitive enzyme-linked immunoassay or a liquid chromatographic assay. Cross-flow microfiltration with 0.22 micron membrane was used to remove the cells. The protein G-rich permeate from the cross-flow microfilter was purified by affinity chromatography using a 5 l column of IgG-Sepharose 6 Fast Flow, which yielded 16-18 g of protein G per column cycle. The pools of purified protein G were concentrated and desalted using ultrafiltration. The salt-free protein G was then lyophilized as bulk product. The overall recovery through the entire process was 50-64%. The analysis of the final product included sodium dodecyl sulfate polyacrylamide gel electrophoresis, UV-visible spectrum, high performance gel filtration, endotoxin level and binding efficiency to human IgG Sepharose.  相似文献   

13.
The cry1I genes from Bacillus thuringiensis are a class of special genes with unique characteristics; they are silent in B. thuringiensis strains but can be over-expressed in Escherichia coli, resulting in a Cry1I-type protein with a molecular mass of approximately 81kDa. Cry1I-type protein is toxic to Lepidoptera larvae. A truncated Cry1Ie protein, IE648, which corresponds to the first 648 amino acids from the N-terminus of Cry1Ie, was purified from E. coli using Ni-NTA affinity isolation, Q-Sepharose Fast Flow chromatography, and Superdex-200 size-exclusion chromatography. It was determined using laboratory bioassays that the purified IE648 protein has good insecticidal activity. Heterologous competitive binding assays show that IE648 does not compete with Cry1Ac for binding to the brush border membrane vesicles of the Asian corn borer and does not compete with Cry1Ac at concentrations below a 500-fold excess of unlabeled Cry1Ac for binding to the peritrophic matrix of the insect. This result implies that IE648 may be a good candidate as part of a multiple-toxin strategy for the potential control of resistance in insect pests. The method of purification reported here is valuable for further research on the structure and function of IE648 and in evaluating the biosafety of this protein within transgenic plants.  相似文献   

14.
将人胱硫醚β-合酶(CBS)基因克隆至质粒pGEX-4T-1中,获得的重组质粒pGEX-4T-1-CBS转入大肠杆菌E.coli Rosetta (DE3)菌株,构建了高效表达CBS的重组菌E.coli Rosetta (pGEX4T-1-CBS)。重组菌在0.1mmol/L的IPTG于30℃诱导16h,可溶性CBS表达量达到28mg/L培养基。将重组菌破碎后上清液经GSTrap Fast Flow亲和层析一步纯化得到CBS融合蛋白,在凝血酶柱上切割缓冲液中加入3%甘油和0.1%CHAPS可以有效抑制酶切后CBS聚沉,酶活性回收率为54.8%,蛋白质产率为15.2mg/L培养基,纯度达到95%,单位酶活为143U/mg,终浓度为1mmol/L的S-腺苷甲硫氨酸(AdoMet)可使CBS单位酶活提高5.1倍,达到735U/mg。同时构建了表达CBS1-413(删除了CBS羧基端调控域138个氨基酸残基)的重组菌E.coli Rosetta (pETDuet-1-CBS1-413),经过一步HisTrap Fast Flow亲和层析,酶活性回收率为74.3%,蛋白质产率为12.8mg/L培养基,纯度达到95%,单位酶活为965U/mg; 还表达和纯化了胱硫醚β-裂解酶(CBL),并在此基础上建立了一种新的CBL偶联的CBS酶活性测定方法。  相似文献   

15.
Production of recombinant serpins in Escherichia coli   总被引:2,自引:0,他引:2  
Expression systems based on Escherichia coli offer fast, cheap, and convenient means for the production of recombinant serpins. Over 30 active serpins from prokaryotic and eukaryotic organisms have been produced in this way, using a variety of vectors, promoters, fusion partners, and host strains. Serpins forming insoluble inclusion bodies in E. coli can generally be solubilized and refolded. Here, we outline the general approaches and procedures to be considered when contemplating the use of E. coli for recombinant serpin production.  相似文献   

16.
Fast protein liquid chromatography (FPLC) with DEAE-Sepharose Fast Flow, PBE-94 and Q-Sepharose Fast Flow columns are applied to the purification of the ferric enterobactin protein receptor (FepA). The apparent single band of FepA on SDS-PAGE is isolated and purified into two proteins with very similar molecular weights. The two proteins are identified to be FepA and ferric citrate protein receptor (FecA) by N-terminus amino acid determination and a computer search with the Gene Bank file. The assay of binding activities of these proteins shows that both FepA and FecA bind ferric enterobactin, with the former having about double the activity of the latter. Competition studies shows that Fe-MECAM is competitively bound to both proteins and that ferric parabactin only slightly competes with [55Fe]ferric enterobactin. It is found that ferrichrome A has no effect on the binding of the receptor proteins with ferric enterobactin.  相似文献   

17.
大肠杆菌高密度发酵表达肠激酶轻链融合蛋白DsbA-rEKL,主要以包涵体形式存在。包涵体经4mol/L尿素和0.5%TritonX-100洗涤,以6mol/L盐酸胍、100mmol/LDTT溶解,在胱氨酸存在下,以脉冲加样方式复性。融合蛋白复性在6mmol/L胱氨酸存在下、脉冲加量0.03mg/mL和复性终蛋白浓度0.3mg/mL为最佳复性方案。复性的融合蛋白加2mmol/LCaCL2后快速自切。经IDA-Sepharose及Q-Sepharose纯化,rEKL纯度可达95%以上,可高效酶切重组瑞特普酶融合蛋白Trx-rPA。实现了大规模生产rEKL,每升发酵液经复性及纯化后,可得rEKL60mg/L以上,使以融合蛋白表达rPA等药用蛋白成为现实。  相似文献   

18.
Since transglutaminase (TGase) have been widely used in industry, mass production of the enzyme is especially necessary. The mature TGase gene from Streptomyces fradiae was cloned into pET21a and overexpressed in Escherichia coli BL21(DE3). The recombinant TGase was formed as inclusion bodies, and its content was as high as 55% of the total protein content. The insoluble fractions were separated from cellular debris by centrifugation and solubilized with 8 M urea. With an on-column refolding procedure based on cation SP Fast Flow chromatography with dual-gradient, the active TGase protein was recovered efficiently from inclusion bodies. The final purified product was 95% pure detected by SDS-PAGE. Under appropriate experimental conditions, the protein yield and specific activity of the TGase were up to 53% and 21 U/mg, respectively. Furthermore, the refolded recombinant protein demonstrated nearly identical ability to polymerized BSA compared with that of native TGase. One hundred and five milligrams of refolded TGase protein was obtained from 3.2g wet weight cells in the 400 ml cell culture.  相似文献   

19.
DsbA (disulfide bond formation protein A) located in the periplasm of Escherichia coli is a disulfide isomerase, which is vital to disulfide bonds formation directly affecting the nascent peptides folding to the correct conformation. In this paper, recombinant DsbA was firstly immobilized onto NHS-activated Sepharose Fast Flow gel. Then Sephadex G-100 gel was sequentially packed on the top of recDsbA Sepharose Fast Flow, and a so-called conjoint chromatography column composed of SEC and immobilized recombinant DsbA was constructed. Denatured lysozyme was applied on the conjoint column. The effect of SEC volume, flow rate, loading amount and volume, pre-equilibrium mode and KCl concentration in the buffer on lysozyme refolding were investigated in detail and the stability of DsbA immobilization was evaluated. Finally the reusability of the conjoint refolding column was also tested. When loading 2.4 mg denatured lysozyme in 0.5 ml solution, the activity recovery reached 92.7% at optimized experimental conditions, and the conjoint column renaturation capacity decreased only 7.7% after six run reuse due to the use of SEC section in the chromatographic refolding process. The conjoint chromatography offers an efficient strategy to refold proteins in vitro with high productivity and column reusability.  相似文献   

20.
The key step in the purification of a deoxyribonuclease (DNase) from extracts of cod (Gadus morhua L.) pyloric caeca, is the selective retention of the enzyme by anion exchange chromatography. The cod DNase purification on Q-Sepharose Fast Flow (Pharmacia) was optimized, using a 60 ml fixed-bed column. In combination with titration curve analysis, we have screened the effect of buffer pHs, feed conductivity and protein loading, on the product recovery and purity. We have developed elution conditions which allow effective separation of the cod DNase from bounded impurities, such as proteinases and nucleic acids. Low levels of these impurities were regarded as essential for the desired product quality. The optimum resolution and maximum purification (ca. 20-fold increase in specific activity) of DNase, was, however, achieved at low protein loading (2.6 mg ml-1 gel), corresponding to less than 4% of the dynamic bed capacity. Scale-up to a 2.5 l pilot scale column (axial flow) and a 0.25 l radial flow column showed that the separation and yield obtained at laboratory scale was retained, and was independent of column geometry and bed height. The implications for a production scale scenario of 100 g of fractionated protein, are also discussed, as well as process hygiene. The optimization described herein adds further knowledge to the treatment of fish waste and the downstream processing of valuable biochemicals from marine raw material.  相似文献   

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