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使芳族代谢物生产接近理论产量的大肠杆菌中心代谢工程

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生物技术通报 1995年第5期 101页

作者：孙雷心

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蓖麻毒素A链与绿色荧光蛋白基因的融合表达和活性测定

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浙江大学学报（医学版） 2005年第3期34卷 201-206页

作者：陈欣虹刘琼詹金彪浙江大学医学院生物化学与分子生物学教研室

　目的:表达蓖麻毒素A链(RTA)与绿色荧光蛋白(GFP)的融合蛋白(GFP-RTA),用于蓖麻毒素A链在细胞内的转运机理研究。方法:采用PCR法从重组质粒pKK233.2-RTA中扩增出蓖麻毒素A链基因,然而将其插入真核表达质粒pEGFPC1,构成GFP-RTA融合基因;... 详细信息

　目的:表达蓖麻毒素A链(RTA)与绿色荧光蛋白(GFP)的融合蛋白(GFP-RTA),用于蓖麻毒素A链在细胞内的转运机理研究。方法:采用PCR法从重组质粒pKK233.2-RTA中扩增出蓖麻毒素A链基因,然而将其插入真核表达质粒pEGFPC1,构成GFP-RTA融合基因;GFP-RTA经PCR扩增,与T载体连接,用内切酶从T-GFP-RTA中切下GFP-RTA片段,插入原核表达质粒pET-28a(+);在BL21(DE3)中表达GFP-RTA融合蛋白,通过金属螯合和层析纯化,MTT法检测融合蛋白的细胞毒性。结果:测序发现插入的融合基因全长序列完全正确,SDS-PAGE鉴定表达产物分子量正确;经诱导表达后,菌体产生绿色荧光,MTT法表明该融合蛋白呈现与RTA相似的细胞毒性。结论:从大肠杆菌表达的蓖麻毒素A链与绿色荧光蛋白的融合蛋白,具有蓖麻毒素A链和绿色荧光蛋白的生物学活性,可用于蓖麻毒素A链在细胞内的转运途径研究。

关键词：蓖麻蛋白蓖麻毒素A链发光蛋白质类重组融合蛋白质类/生物合成基因表达大肠杆菌/代谢聚合酶链反应

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重组IFN-α-2b-BCG对人PBMC的TLR4表达调节及其抗肿瘤作用的研究

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实用肿瘤杂志 2010年第2期25卷 141-145页

作者：孙二琳王玉叶韩瑞发孙岩天津市泌尿外科研究所天津300211

目的了解重组卡介苗(BCG)和野生BCG对人外周单核细胞(hPBMC)上TLR的调节差异,揭示hIFN-α-2b对hPBMC上TLR4的表达是否具有协同增强效应,以及BCG介导免疫细胞活化效应的新机制。方法比较研究重组hIFN-α-2b-BCG和野生型BCG,及其上清和等... 详细信息

目的了解重组卡介苗(BCG)和野生BCG对人外周单核细胞(hPBMC)上TLR的调节差异,揭示hIFN-α-2b对hPBMC上TLR4的表达是否具有协同增强效应,以及BCG介导免疫细胞活化效应的新机制。方法比较研究重组hIFN-α-2b-BCG和野生型BCG,及其上清和等量干扰素对TLR4表达的调节,以及表达TLR4淋巴细胞的肿瘤杀伤效应。以淋巴细胞作为空白对照,应用流式细胞仪对TLR4进行检测,MTT法检测肿瘤细胞杀伤效应。结果证实了重组BCG和野生BCG对hPBMC的TLR4的表达均有正调节及抗肿瘤效应;重组BCG对hPBMC的TLR4表达的调节优于野生BCG(P<0.05);重组BCG上清和外源性等量干扰素对TLR4表达皆有正调节作用,但差别无统计学意义;重组BCG上清与野生BCG上清相比,对hPBMC诱导TLR4表达差别显著(P<0.05);与野生BCG组相比,重组BCG组表达TLR4的hPBMC杀伤肿瘤细胞的效应显著增强(P<0.05)。结论重组BCG的脂多糖和持续分泌的hIFN-α-2b对hPBMC的TLR4表达均有正调节作用,并加强表达TLR4淋巴细胞的细胞介导的抗肿瘤效应。

关键词：干扰素α/代谢受体,细胞表面膜糖蛋白类/代谢卡介苗/治疗应用淋巴细胞大肠杆菌/代谢分枝杆菌,牛/代谢卡介苗/生物合成重组,遗传肿瘤/治疗

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GAS TRANSFER IN MICROGRAVITY FERMENTATIONS

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ACTA ASTRONAUTICA 1986年第3期13卷 105-117页

作者： THOMPSON, BG WARD, D Department of Biotechnology Alberta Research Council Edmonton Canada.

Fermentations performed under microgravity conditions may be used in future long duration space missions for recycling expandable life support materials. These fermentations will differ from similar fermentations perf... 详细信息

关键词：生物量生物反应器计算机模拟生态系统封闭大肠杆菌/生长和发育大肠杆菌/代谢发酵气体/化学生命保障系统/仪器和设备宇宙飞行/仪器和设备废弃物处理/仪器和设备废弃物处理/方法失重

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Construction and expression of functional multi-domain polypeptides in Escherichia coli:: expression of the Neurospora crassa metallothionein gene

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LETTERS IN APPLIED MICROBIOLOGY 2000年第2期30卷 161-166页

作者： Mauro, JM Pazirandeh, M USN Res Lab Ctr Biomol Sci & Engn Washington DC 20375 USA

A system for the construction of polymeric peptides in Escherichia coli was utilized to prepare a library of plasmids coding for tandem repeats of the Neurospora crassa metallothionein gene. Selected oligomeric metallothionein clones were expressed and target-ed to the periplasm as a fusion with the maltose-binding protein. Bacterial cells harbouring the expressed oligopeptides were characterized for their ability to bind Cd-109(2+). The metal-binding ability was enhanced for all the oligomeric constructs tested and, in the best case, a 6.5-fold increased capacity for metal uptake was achieved with cells expressing a tandem 9-mer in comparison with cells expressing a monomer. Plateauing of the metal uptake ability occurred at between six and nine tandem repeats, possibly due to a combination of lowered translation levels, inefficient export and prematurely terminated translation products. The overall enhancement of the heavy metal removal capacity was approximately 65-fold relative to non-recombinant cells. The use of this strategy for the design and expression of de novo polypeptides containing multiple functional domains for use in bioremediation is discussed.

关键词： ATP结合匣式转运子氨基酸序列碱基序列镉/代谢载体蛋白质类/代谢大肠杆菌/化学大肠杆菌/遗传学大肠杆菌/代谢大肠杆菌蛋白质类麦芽糖结合蛋白质类金属硫蛋白/生物合成金属硫蛋白/化学金属硫蛋白/遗传学金属硫蛋白/代谢分子序列数据单糖转运蛋白质类粗糙链孢霉/遗传学粗糙链孢霉/代谢寡肽类/生物合成寡肽类/代谢质粒/遗传学蛋白质工程重组融合蛋白质类/生物合成串联重复序列/遗传学

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Signal peptide determinants of SecA binding and stimulation of ATPase activity

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第14期275卷 10154-10159页

作者： Wang, LG Miller, A Kendall, DA Univ Connecticut Dept Mol & Cell Biol Storrs CT 06269 USA

A signal peptide is required for entry of a preprotein into the secretory pathway, but how it functions in concert with the other transport components is unknown. In Escherichia coli, SecA is a key component of the translocation machinery found in the cytoplasm and at membrane translocation sites. Synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and three sets of model signal sequences varying in hydrophobicity and amino-terminal charge were generated. These were used to establish the requirements for interaction with SecA, Binding to SecA, modulation of SecA conformations sensitive to protease, and stimulation of SecA-Lipid ATPase activity occur with functional signal sequences but not with transport-incompetent ones. The extent of SecA interaction is directly related to the hydrophobicity of the signal peptide core region. For signal peptides of moderate hydrophobicity, stimulation of the SecA-lipid ATPase activity is also dependent on amino-terminal charge. The results demonstrate unequivocally that the signal peptide, in the absence of the mature protein, interacts with SecA in aqueous solution and in a lipid bilayer. We show a clear parallel between the hierarchy of signal peptide characteristics that promote interaction with SecA in vitro and the hierarchy of those observed for function in vivo.

关键词：腺苷三磷酸酶类/化学腺苷三磷酸酶类/代谢腺苷酰亚氨二磷酸/药理学氨基酸序列氨基酸取代细菌蛋白质类/化学细菌蛋白质类/代谢载体蛋白质类/化学载体蛋白质类/代谢大肠杆菌/代谢大肠杆菌蛋白质类动力学脂质体膜转运蛋白质类分子序列数据蛋白质构象蛋白分选信号/化学合成蛋白分选信号/化学蛋白分选信号/代谢丝氨酸内肽酶类

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The ends of tropomyosin are major determinants of actin affinity and myosin subfragment 1-induced binding to F-actin in the open state

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BIOCHEMISTRY 1999年第48期38卷 15885-15892页

作者： Moraczewska, J Nicholson-Flynn, K Hitchcock-DeGregori, SE Univ Med & Dent New Jersey Robert Wood Johnson Med Sch Dept Neurosci & Cell Biol Piscataway NJ 08854 USA

Tropomyosin (TM) is thought to exist in equilibrium between two states on F-actin, closed and open [Geeves, M. A., and Lehrer, S. S. (1994) Biophys. J. 67, 273-282]. Myosin shifts the equilibrium to the open state in which myosin binds strongly and develops force. Tropomyosin isoforms, that primarily differ in their N- and C-terminal sequences, have different equilibria between the closed and open states. The aim of the research is to understand how the alternate ends of TM affect cooperative actin binding and the relationship between actin affinity and the cooperativity with which myosin S1 promotes binding of TM to actin in the open state. A series of rat alpha-tropomyosin variants was expressed in Escherichia coli that are identical except for the ends, which are encoded by exons la or Ib and exons 9a, 9c or 9d. Both the N- and C-terminal sequences, and the particular combination within a TM molecule, determine actin affinity. Compared to tropomyosins with an exon la-encoded N-terminus, found in long isoforms, the exon Ib-encoded sequence, expressed in 247-residue nonmuscle tropomyosins, increases actin affinity in tropomyosins expressing 9a or 9d but has little effect with 9c, a brain-specific exon. The relative actin affinities, in decreasing order, are 1b9d > 1b9a, acetylated 1a9a > 1a9d much greater than 1a9a greater than or equal to 1a9c congruent to 1b9c. Myosin S1 greatly increases the affinity of all tropomyosin variants for actin. In this, the actin affinity is the primary factor in the cooperativity with which myosin S1 induces TM binding to actin in the open state;generally, the higher the actin affinity, the lower the occupancy by myosin required to saturate the actin with tropomyosin: 1b9d > 1a9d > 1b9a greater than or equal to acetylated 1a9a > 1a9a > la9c congruent to 1b9c.

关键词：肌动蛋白类/化学氨基酸序列鸡 DNA 互补/代谢大肠杆菌/遗传学大肠杆菌/代谢基因表达分子序列数据肌球蛋白亚碎片/药理学肌球蛋白类/药理学蛋白质结合/药物作用蛋白质构象蛋白质折叠原肌球蛋白/生物合成原肌球蛋白/化学原肌球蛋白/遗传学肌钙蛋白/化学动物大鼠

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The quiescent-cell expression system for protein synthesis in Escherichia coli

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第6期65卷 2710-2715页

作者： Rowe, DCD Summers, DK Univ Cambridge Dept Genet Cambridge CB2 3EH England

The quiescent-cell expression system is a radical alternative to conventional fermentation for protein overproduction in Escherichia coli. It is dependent on the controlled overexpression of a small RNA called Red in hns mutant, strains to generate nongrowing, quiescent cells which are not nutrient limited. Quiescent cells no longer produce biomass and have their metabolic resources channelled toward the expression of plasmid-based genes. The. biosynthetic capacity of the system is demonstrated by its ability to express chloramphenicol acetyltransferase to more than. 40% of total cell protein. Quiescent cells may provide an ideal environment for the expression of toxic as well as benign proteins.

关键词：细菌蛋白质类/生物合成氯霉素O-乙酰转移酶/生物合成 DNA结合蛋白质类/遗传学 DNA结合蛋白质类/代谢大肠杆菌/遗传学大肠杆菌/生长和发育大肠杆菌/代谢基因表达调控细菌乳糖操纵子甲硫氨酸/代谢质粒 RNA 细菌/遗传学 RNA 细菌/代谢硫放射性同位素/代谢 β半乳糖苷酶类/生物合成

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Functions of the σ⁵⁴ region I in Trans and implications for transcription activation

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第36期274卷 25285-25290页

作者： Gallegos, MT Cannon, WV Buck, M Univ London Imperial Coll Sci Technol & Med Dept Biol London SW7 2AZ England

Control of transcription frequently involves the direct interaction of activators with RNA polymerase, In bacteria, the formation of stable open promoter complexes by the sigma(54) RNA polymerase is critically dependent on sigma(54) amino Region I sequences. Their presence correlates with activator dependence, and removal allows the holoenzyme to engage productively with melted DNA independently of the activator. Using purified Region I sequences and holoenzymes containing full-length or Region I-deleted sigma(54), we have explored the involvement of Region I in transcription activation, Results show that Region I in trans inhibits a reversible conformational change in the holoenzyme believed to be polymerase isomerization. Evidence is presented indicating that the holoenzyme land not the promoter DNA per se) is one interacting target used by Region I in preventing polymerase isomerization, Activator overcomes this inhibition in a reaction requiring nucleotide hydrolysis. Region I in trans is able to inhibit activated transcription by the holoenzyme containing full-length sigma(54). Inhibition appeared to be noncompetitive with respect to the activator, suggesting that a direct activator interaction occurs with parts of the holoenzyme outside Region I. Stabilization of isomerized holoenzyme bound to melted DNA by Region I in trans occurs largely independently of the initiating nucleotide, suggesting a role for Region I in maintaining the open complex.

关键词：细菌蛋白质类/遗传学细菌蛋白质类/代谢 DNA结合蛋白质类指导DNA的RNA聚合酶类/遗传学指导DNA的RNA聚合酶类/代谢大肠杆菌/遗传学大肠杆菌/代谢大肠杆菌蛋白质类基因表达调控细菌 RNA聚合酶∑54 ∑因子/遗传学 ∑因子/代谢转录遗传转录激活

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The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil)

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第1期88卷 170-175页

作者： Cox, SD Mann, CM Markham, JL Bell, HC Gustafson, JE Warmington, JR Wyllie, SG Univ Western Sydney Ctr Biostruct & Biomol Res Hawkesbury NSW Australia Australian Tea Tree Oil Res Inst Lismore NSW Australia Genet Biotechnol Bentley WA Australia

The essential oil of Melaleuca alternifolia (tea tree) exhibits broad-spectrum antimicrobial activity. Its mode of action against the Gram-negative bacterium Escherichia coli AG100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325, and the yeast Candida albicans has been investigated using a range of methods. We report that exposing these organisms to minimum inhibitory and minimum bactericidal/fungicidal concentrations of tea tree oil inhibited respiration and increased the permeability of bacterial cytoplasmic and yeast plasma membranes as indicated by uptake of propidium iodide. In the case of E. coli and Staph. aureus, tea tree oil also caused potassium ion leakage. Differences in the susceptibility of the test organisms to tea tree oil were also observed and these are interpreted in terms of variations in the rate of monoterpene penetration through cell wall and cell membrane structures. The ability of tea tree oil to disrupt the permeability barrier of cell membrane structures and the accompanying loss of chemiosmotic control is the most likely source of its lethal action at minimum inhibitory levels.

关键词：抗菌药抗感染药/药理学白色念珠菌/药物作用白色念珠菌/生长和发育白色念珠菌/代谢细胞膜通透性细胞呼吸/生理学大肠杆菌/药物作用大肠杆菌/生长和发育大肠杆菌/代谢微生物敏感性试验钾/代谢普罗匹定/代谢金黄色葡萄球菌/药物作用金黄色葡萄球菌/生长和发育金黄色葡萄球菌/代谢茶树油/药理学

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