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1.
SUMO蛋白酶(Ulp1)是切割小分子泛素修饰(SUMO)融合蛋白获得天然N端靶蛋白的一种工具酶,具有酶切效率高、特异性好等优点。但现有市售SUMO蛋白酶Ulp1价格昂贵、操作复杂,限制了SUMO融合体系的运用。利用基因工程技术,合成基因ulp1(Leu403-Lys621),并在N端和C端加入多聚组氨酸标签(His_6),构建重组表达载体psv T7-ulp1,将重组质粒转入大肠杆菌BL21(DE3)和BL21 trx B(DE3)中。经过高通量筛选技术快速确定最优的表达条件为采用BL21(DE3)作为表达宿主,转接后7h加入IPTG,IPTG的终浓度为0.1mmol/L,诱导时间为16h,最终蛋白质表达量占菌体总蛋白质量的34.5%,重组蛋白Ulp1的表达量为190mg/L,通过Ni-NTA一步纯化即可得到纯度95%以上的Ulp1。通过酶切反应,测定酶活为5.19U/μl,比酶活为5.23×10~4U/mg,是先前报道比酶活的1.87倍,通过酶活动力学分析,Ulp1的表观米氏常数K_m=0.359g/L,V_m=5.10μg/(ml·min)。将SUMO融合表达体系用于单链抗体(single-chain antibody fragment,scFv)的表达,得到可溶的SUMO-scFv融合蛋白,使用表达的Ulp1进行酶切并纯化,获得纯度高于90%且N端不含多余氨基酸的scFv,操作步骤简单,显著改善了scFv在大肠杆菌中难以高效可溶性表达纯化的现状。 相似文献
2.
《生物技术通讯》2018,(5)
目的:在大肠杆菌中表达并纯化人铜锌超氧化物歧化酶(HuSOD1)。方法:合成HuSOD1编码基因,PCR扩增后连入pMAL-p5x质粒构建融合表达载体,转化大肠杆菌BL21(DE3)感受态,IPTG诱导表达,NBT法测定HuSOD1酶活,利用麦芽糖结合蛋白亲和层析柱纯化MBP-HuSOD1融合蛋白,经因子Ⅹa酶切及分子筛柱层析纯化HuSOD1蛋白。结果:构建了pMAL-p5x-HuSOD1表达载体,在大肠杆菌中实现了高表达,目的蛋白占全菌蛋白的30%,其中可溶性表达占63%,具有超氧化物歧化酶活性;通过亲和层析纯化得到纯度大于95%的融合蛋白MBP-HuSOD1,经因子Ⅹa酶切后纯化得到纯度约90%的HuSOD1蛋白。结论:在大肠杆菌中表达并纯化获得有活性的MBP-HuSOD1,经进一步酶切、纯化后得到HuSOD1。 相似文献
3.
将来源于采采蝇的TTI基因序列改造成大肠杆菌偏爱密码子,利用重组PCR方法获得TTI目的基因片段,在大肠杆菌中得到高效表达。经纯化获得了纯度高于98%的融合蛋白,建立了酶活测定方法。实验证明融合蛋白具有抑制凝血酶的活性。当凝血酶浓度为10U/ml,纯化的融合TTI体积为10 l,底物浓度为250 mol/L,融合蛋白对凝血酶的抑制率为73%,确定反应类型为竞争性抑制,Ki为35 mol/L。 相似文献
4.
《生物技术通讯》2020,(1)
目的:在乳酸乳球菌中重组表达乙醛脱氢酶(ALDH)。方法:合成毕赤酵母ALDH基因,PCR扩增后通过重组构建pNZ8048-ALDH表达载体,电转至乳酸乳球菌NZ9000感受态,Nisin诱导表达后经Ni柱亲和层析纯化ALDH蛋白,比色法测定酶活。结果:构建了pNZ8048-ALDH表达载体,在乳酸乳球菌NZ9000中实现了ALDH的重组表达,目的蛋白占全菌蛋白的17.2%,其中可溶性表达比例为53%,重组菌株ALDH活力为0.638 U/mL,亲和层析纯化蛋白纯度约70%,比活为0.48 U/mg。结论:在乳酸乳球菌中表达并纯化获得了有活性的ALDH。 相似文献
5.
《生物技术通报》2016,(11)
旨在大肠杆菌中可溶表达重组人神经生长因子(Recombinant humanβnerve growth factor,rhβNGF),并对表达产物进行分离纯化和生物学活性鉴定。成功扩增h NGFβ亚基基因,将其克隆入pMAL-c2X表达载体,构建了hβNGF-MBP的大肠杆菌表达体系并进行诱导表达,表达产物经纯化后以Factor Xa酶切去除麦牙糖结合蛋白(MBP),Western blot鉴定后以TF-1细胞法检测生物学活性。结果显示,pMAL-c2X-hβNGF经酶切和测序证实构建正确,25℃、180 r/min、0.5 mmol/L IPTG诱导下可溶表达hβNGF-MBP融合蛋白。hβNGF-MBP经Factor Xa酶切后可去除MBP标签,SDS-PAGE分析纯化的hβNGF位于13 k D左右,纯度可达95%。Western blot鉴定为hβNGF,结果表明,比活约为1×10~6 U/mg。在大肠杆菌中成功可溶表达hβNGF,并具有较高的生物学活性。 相似文献
6.
核酸酶在生物工程领域有着重要的应用价值。本研究在优化北极虾核酸酶(Shrimp nuclease,SNU)基因序列的基础上,构建SNU的毕赤酵母分泌表达载体SNU-p PICZαA并转化酵母,以高拷贝整合转化子为基础,优化酶表达的条件,并对该酶的催化特性进行分析,结果显示SNU可在毕赤酵母SMD1168H中高效分泌表达,最佳诱导表达条件为:培养基BMMY p H 6.0,甲醇浓度为1%,诱导时间为72 h,诱导后粗酶液比活力为1.4×10~5 U/m L。经过DEAE Sephadex阴离子交换层析可纯化获得高纯度的目标蛋白,每升菌液可纯化15 mg目标蛋白,比活力达到6.291×10~6 U/mg,该酶表观分子量为50 k Da,PNGase F酶切证实该酶存在糖基化现象。二价金属离子Ca~(2+)、Mn~(2+)、Co~(2+)、Mg~(2+)及还原剂DTT、β-ME能显著地提高其水解活性,但Zn~(2+)、Cu~(2+)、SDS、高浓度Na Cl抑制该酶的活性,SNU为Ca~(2+)/Mg~(2+)依赖型核酸酶。70℃处理10 min可使该酶不可逆的失活。 相似文献
7.
目前有关限制性内切酶NotⅠ的性质特征及功能机制等方面的研究日渐增多,但商品化NotⅠ及某些限制性内切酶的价格依然居高不下,其主要原因在于表达量低、提纯程序繁琐、得率低等问题的存在。为探索限制性内切酶NotⅠ提纯的新工艺,从豚鼠耳炎诺卡菌(Nocardia otitidis-caviarum)中克隆出限制性内切酶NotⅠ的基因并使之在大肠杆菌中高效表达。首先将由成团肠杆菌(Enterobacter agglomerans)中克隆所得甲基化酶EagⅠM(EagⅠ methylase gene)基因连接到pBR322载体上,转化大肠杆菌ER2566,将豚鼠耳炎诺卡菌中克隆所得的限制性内切酶NotⅠR(NotⅠrestriction endonuclease gene)基因连接到表达载体pACYC184-PT7上,将此重组质粒转化到上述已转入甲基化重组质粒pBR322-EagⅠM的ER2566中,构建成NotⅠ蛋白表达菌ER2566 。重组工程菌经IPTG诱导可表达限制性内切酶NotⅠ,并对诱导条件进行优化使之以可溶形式高效表达。应用KTA purifier 100蛋白纯化系统,对纯化工艺进行创新,通过DEAE Sephrose FF离子交换层析、phenyl HP疏水层析和Superdex 75 10/300 GL分子筛层析对蛋白进行提纯。纯化后NotⅠ经酶活力及纯度鉴定,其比活力为1.37×106U/mg,提纯35倍,得率为17.8%,产量达9.8×106 Units /g wet cell,提纯时间缩减为原来的1/10,在产量和效率上较以前报道均有很大提高。该纯化工艺的新方法,为实验室制备及工业化生产Ⅱ型限制性内切酶提供了进一步的借鉴。且该酶的成功获得为后续研究提供了材料,为更多新发现内切酶的成功克隆提供了参考。 相似文献
8.
目前有关限制性内切酶Not I的性质特征及功能机制等方面的研究日渐增多,但商品化Not Ⅰ及某些限制性内切酶的价格依然居高不下,其主要原因在于表达量低、提纯程序繁琐、得率低等问题的存在.为探索限制性内切酶Not Ⅰ提纯的新工艺,从豚鼠耳炎诺卡菌(Nocardia otitidiscaviarum)中克隆出限制性内切酶Not Ⅰ的基因并使之在大肠杆菌中高效表达.首先将由成团肠杆菌(Enterobacter agglomerans)中克隆所得甲基化酶Eag I M(Eag I methylase gene)基因连接到pBR322载体上,转化大肠杆菌ER2566,将豚鼠耳炎诺卡菌中克隆所得的限制性内切酶Not IR(Not I restriction endonuclease gene)基因连接到表达载体pACYC184-PT7上,将此重组质粒转化到上述已转入甲基化重组质粒pBR322-Eag I M的ER2566中,构建成Not I蛋白表达菌ER2566[pBR322-Eag I M,pACYC184-PT7-Not I R].重组工程菌经IPTG诱导可表达限制性内切酶Not Ⅰ,并对诱导条件进行优化使之以可溶形式高效表达.应用(A)KTA purifier 100蛋白纯化系统,对纯化工艺进行创新,通过DEAE Sephrose FF离子交换层析、phenyl HP疏水层析和Superdex 75 10/300GL分子筛层析对蛋白进行提纯.纯化后Not Ⅰ经酶活力及纯度鉴定,其比活力为1.37 × 10(6)U/mg,提纯35倍,得率为17.8%,产量达9.8×10(6)Units /g wet cell,提纯时间缩减为原来的1/10,在产量和效率上较以前报道均有很大提高.该纯化工艺的新方法,为实验室制备及工业化生产Ⅱ型限制性内切酶提供了进一步的借鉴.且该酶的成功获得为后续研究提供了材料,为更多新发现内切酶的成功克隆提供了参考. 相似文献
9.
【目的】在原核表达系统中实现人肠激酶轻链(Human enterokinase light chain,hEKL)的表达和纯化。【方法】通过PCR扩增得到编码hEKL的基因片段,利用基因重组技术构建原核表达质粒pMAL-s-hEKL,在Escherichia coli中进行诱导表达,菌体经超声破碎后利用Amylose亲和柱对目标蛋白进行纯化,并利用Tricine SDS-PAGE检测酶的切割活性。【结果】目的基因能够以可溶形式表达,每升发酵液可纯化得到40 mg纯度在97%以上的MBP-hEKL蛋白,活性检测表明该酶可以对含有肠激酶识别序列的蛋白进行特异性切割,酶活力达到6.0×105U/mol。 相似文献
10.
《生物技术》2015,(5)
[目的]重组融合蛋白Trx-IFN-CSP的肠激酶酶切及纯化研究。[方法]选用国产肠激酶,在不同反应条件下酶切重组融合蛋白,15%的SDS-PAGE检测切割产物,并利用Ni2+亲和层析色谱联合肝素亲和层析色谱对酶切产物进行分离纯化。[结果]0.8 U的肠激酶在温度为10℃下酶切72 h能够完全裂解50μg融合蛋白。酶切产物经亲和层析色谱纯化,可获得电泳纯达99%的目的蛋白。[结论]重组融合蛋白Trx-IFN-CSP经肠激酶酶切及纯化后,可获得纯度达99%的肝靶向干扰素IFN-CSP单体,为进一步研究肝靶向干扰素的生物学活性及产业化开发奠定了基础,也为融合蛋白后处理工艺提供了技术借鉴。 相似文献
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Pushkar Shejwalkar 《Inorganica chimica acta》2011,366(1):209-218
New sulfur derivatives of phosphoramidite ligands were synthesized and the impact of the sulfur unit on the spectroscopic properties of their rhodium and iridium complexes was investigated. The new ligands Bn2NPSCH2CH2Sa(P-Sa) (Bn = benzyl, 4), Bn2NPSCHCHSa(CH2)3CaH2(P-Sa)(Ca-Sa) (6) and Bn2NP(4-XC6H4OMe)2 (X = S, 7a; X = O, 7b) were converted to the rhodium and iridium complexes trans-[Rh(CO)Cl(L)2] (L = 4, 6, 7), [RhCl(COD)(L)] (L = 4, 6, 7), [IrCl(COD)(7a)] and [IrCl2Cp∗(6)]. For comparison, some phosphoramidite complexes of these formulations also were synthesized. The new metal complexes were spectroscopically analyzed. For the carbonyl complexes, the νCO IR stretching frequencies were lower than for the corresponding phosphite and phosphoramidite ligands. The 1JPRh coupling constants for the rhodium complexes with the new ligands were also smaller than for the respective phosphoramidite and phosphite complexes. Finally, the 1JPSe coupling constants of the selenides of the new ligands were lower than those of the phosphoramidite ligands but higher than for PPh3. The spectroscopic data reveal that the new thio ligands 4, 6 and 7a are more electron donating than phosphites and phosphoramidites but less electron donating than PPh3. 相似文献
13.
Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes. 相似文献
14.
Hand JW 《Progress in biophysics and molecular biology》2007,93(1-3):192-194
The first and second sessions of the Workshop focussed on the basics of ultrasound and infrasound, their applications in both industry and medicine, and metrology and protection standards for ultrasound applications. 相似文献
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Gonzalez-Reimers E Rodriguez-Moreno F Martinez-Riera A Mas-Pascual A Delgado-Ureta E Galindo-Martin L Arnay-de la Rosa M Santolaria-Fernandez F 《Biological trace element research》1999,68(1):41-49
To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively
coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women,
ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred
in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted
that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined
to date.
Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in
women than in men, whereas their content in the external iliac artery was lower in women than in men. 相似文献
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J. Morley M.A. Bray R.W. Jones D.H. Nugteren D.A. van Dorp 《Prostaglandins & other lipid mediators》1979,17(5):729-736
The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection.The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemic leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE2, PGD2, PGF2α, TXB2 and 6-keto-PGF1α. In all types studied PGE2 and TXB2 were the major products formed. The identification of PGE2 and TXB2 was confirmed by GC/MS with multiple ion monitoring.The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology. 相似文献
19.
Allergic asthma can be precipitated by many factors. For the atopic person, fungus, pollen, dust mites, cockroach antigens, and diesel exhaust are all agents that may trigger an allergic attack. Cytokines and chemokines are integral mediators of fungal asthma. From the earliest time points, they recruit and activate the cells required for the clearance of fungus as well as being critical factors involved in the immunopathology of this disease. In the final analysis, it is clear that these mediators can act to the benefit or the detriment of the host. 相似文献
20.
Sandra M. Vaz 《Archives of biochemistry and biophysics》2009,484(2):127-431
In spite of the many studies on protein modifications by reactive species, knowledge about the products resulting from the oxidation of protein-aromatic residues, including protein-derived radicals and their stable products, remains limited. Here, we compared the oxidative modifications promoted by peroxynitrite and myeloperoxidase/hydrogen peroxide/nitrite in two model proteins, ribonuclease (6Tyr) and lysozyme (3Tyr/6Trp). The formation of protein-derived radicals and products was higher at pH 5.4 and 7.4 for myeloperoxidase and peroxynitrite, respectively. The main product was 3-nitro-Tyr for both proteins and oxidants. Lysozyme rendered similar yields of nitro-Trp, particularly when oxidized by peroxynitrite. Hydroxylated and dimerized products of Trp and Tyr were also produced, but in lower yields. Localization of the main modified residues indicates that peroxynitrite decomposes to radicals within the proteins behaving less specifically than myeloperoxidase. Nitrogen dioxide is emphasized as an important protein modifier. 相似文献