基于GC-MS的阿维链霉菌代谢物组学研究方法的建立

基于GC-MS分析平台,建立了阿维链霉菌代谢物组样品制备过程中的菌体分离、细胞清洗、细胞淬灭及代谢物提取条件,并对阿维菌素高产菌株和野生菌株胞内代谢物组进行了初步分析。采用"冷冻离心-细胞清洗(3次)-液氮淬灭(5min)-珠磨破壁(3个循环,每个循环48 s)-提取(氯仿∶乙醇∶水=2∶2∶1)"的方法,可以定性和定量检测阿维链霉菌胞内包括氨基酸、有机酸、脂肪酸、糖类等59种差异化合物。建立的方法能够有效地用于阿维链霉菌胞内代谢物的分析。     

阿维链霉菌NRRL8165中bldAa的克隆及其对形态分化与阿维菌素合成的影响

bldA编码天蓝色链霉菌中唯一有效识别UUA亮氨酸密码的tRNA(Leu)UUA。通过构建阿维链霉菌NRRL8165基因组亚文库,筛选得到含有阿维链霉菌bldAa及其侧翼序列的克隆。利用λRED介导的PCRtargeting技术构建了bldAa的基因置换质粒pHL358,将其跨属接合转移进入阿维链霉菌NRRL8165,筛选得到bldAa基因置换菌株TW10。TW10表现为光秃表型,表明bldAa调控阿维链霉菌的形态分化。摇瓶发酵TW10菌株并对发酵产物进行HPLC分析,发现TW10菌株均不合成阿维菌素组分,提示阿维菌素的合成受bldAa调控;考察阿维菌素生物合成基因簇,其中aveA3和aveR含有TTA密码,它们的翻译可能受bldAa调控,与实验结果一致。     

阿维链霉菌NRRL8165中bldAA的克隆及其对形态分化与阿维菌素合成的影响

bldA编码天蓝色链霉菌中唯一有效识别UUA亮氨酸密码的tRNA（Leu）UUA。通过构建阿维链霉菌NRRL8165基因组亚文库,筛选得到含有阿维链霉菌bldA。及其侧翼序列的克隆。利用λRED介导的PCR targeting技术构建了bldA。的基因置换质粒pHL358,将其跨属接合转移进入阿维链霉菌NRRL8165,筛选得到bldA。基因置换菌株TW10。TW10表现为光秃表型,表明bldA。调控阿维链霉菌的形态分化。摇瓶发酵TW10菌株并对发酵产物进行HPLC分析,发现TW10菌株均不合成阿维菌素组分,提示阿维菌素的合成受bldA。调控;考察阿维菌素生物合成基因簇,其中areA3和aveR含有TTA密码,它们的翻译可能受bldA。调控,与实验结果一致。     

林肯链霉菌谷氨酰胺合成酶活力调节的研究

对不同氮源生长条件下林肯链霉菌无细胞粗提液中谷氨酰胺合成酶 (GS)的研究结果表明 ,高浓度NH+4阻遏了GS的生物合成。从不同氮源生长条件下林肯链霉菌中分离纯化了GS ,其性质没有差别。以受腺苷化调节的产气克雷伯氏菌GS作对照 ,林肯链霉菌GS没有明显的氨休克作用 ,经蛇毒磷酸二酯酶处理后 ,其活力没有变化。这些结果都说明林肯链霉菌GS不存在腺苷化共价修饰这一调节方式。反馈抑制作用是林肯链霉菌GS的一种重要的调节方式 ,这种抑制作用是以累积的方式进行的 ,这表明各种抑制剂对GS作用位点不同 ,各种抑制剂对GS的抑制作用是相互独立的。由此推测 ,林肯链霉菌GS是一种变构酶。     

间α胰蛋白酶抑制剂与胞外间质

间α胰蛋白酶抑制剂(ITI)家族是在人和动物的血液中广泛存在的一类蛋白质.ITI由一条硫酸软骨素糖链将三条蛋白质肽链共价地结合在一起,其中两条肽链与硫酸软骨素链以氨基酸的羧基与糖链上的羟基形成酯键的形式相连.在细胞外间质中, 这种酯键可以转移到透明质酸糖链上, 进而调控细胞外间质的大小和细胞的性质.另一条肽链具有Kunitz型蛋白酶抑制剂的结构, 作为一种临床应用的药物可以从尿中分离到.     

透明颤菌血红蛋白在肉桂地链霉菌中的表达对基因细胞生长及抗生素合成的影响

肉桂地链霉菌（S.cinnamonensis)是莫能菌素（Monensin)的产生菌,大肠杆菌－链霉菌穿梭表达载体pHZ1252中的透明颤菌血红蛋白基因（vhb)位于硫链丝菌素诱导启动子ＰtipA之下,它在肉桂地链霉菌中的结构不稳定,,发生了重组缺失,缺失的片段包括大肠杆菌质粒部分vhb基因。但来自阿维链霉菌(S.avermitilis)中缺失了大肠杆菌质粒部分却保留了完整的vhb基因及tipA启动子的pHZ1252,可在肉桂地链霉菌中稳定复制,不再发生缺失,经硫链丝菌素诱导表达出了有生物活性的ＶＨb蛋白,摇瓶发酵实验证明,ＶＨb蛋白在氧限条件下可明显促进肉桂地链霉菌的菌体生长和抗生素合成。     

山黧豆叶片蛋白质双向电泳技术的建立

以山黧豆叶片为材料,比较分析了蛋白质的不同提取方法,在此基础上着重于样品制备。对IPG胶条的选择,第一向等电聚焦和第二向SDS-聚丙烯酰胺凝胶电泳的电泳程序及参数、染色方法等相关技术进行了比较和条件优化。结果显示：采用TCA-丙酮沉淀法提取蛋白质,裂解液中加入Tris-base作为蛋白酶抑制剂,等电聚焦电泳时延长低电压的电泳时间（30V、12h,500V、1h,1000V、2h）以促进盐离子泳出的方法对山黧豆叶片蛋白质进行双向电泳,并用考马斯亮蓝和银染复合染色法进行凝胶染色,能够获得蛋白点清晰的双向电泳图谱,说明用优化后的方法建立起的山黧豆叶片蛋白质双向电泳技术,蛋白质样品制备质量好,电泳分辨率高,完全适合于进一步的蛋白质组学研究。     

阿维链霉菌来源的磷脂酰丝氨酸合成酶基因的克隆及其在毕氏酵母中的异源表达

本文旨在构建阿维链霉菌(Streptomyces avermitilis)来源的磷脂酰丝氨酸合成酶基因(pss)的重组质粒,研究其在毕氏酵母中的异源分泌型表达。利用PCR技术克隆阿维链霉菌来源的pss基因,再通过电转化方法将重组质粒pOG-01转入毕氏酵母KM71中,构建重组工程菌KP1。实验结果表明,阿维链霉菌来源的磷酯酰丝氨酸合成酶基因在毕氏酵母KM71中成功表达,2 mL菌体上清催化50 mmol/L卵磷脂,转酯反应的转化率为58%,酶活为4.83 U/mL。     

无机盐对阿维链霉菌接合转移及异源表达放线紫红素的影响

摘要：【目的】阿维链霉菌可作为异源表达抗生素生物合成基因簇的良好宿主,但是需要优化含有大片段DNA质粒的接合转移效率。【方法】我们选取MgCl2、NaCl、Ca(NO3)2 和CaCl2等4种无机盐,在0－200 mmol/L浓度范围内分别研究其对大质粒向阿维链霉菌接合转移的影响,再设计完全随机试验筛选最佳条件。【结果】CaCl2对阿维链霉菌接合转移有极明显的促进作用,MgCl2也有一定提高作用。通过完全随机试验筛选出最佳的CaCl2和MgCl2浓度组合,使大质粒的接合转移效率提高11倍。同时,本研究还发现阿维链霉菌异源表达放线紫红素的最适培养基,成功表达放线紫红素。【结论】特定无机盐对阿维链霉菌接合转移效率有明显提高作用,并且能促进放线紫红素在阿维链霉菌中的表达。     

透明颤菌血红蛋白基因在阿维链霉菌中的表达

将含有自身启动子的透明颤菌血红蛋白基因（ ｖｈｂ） 克隆至大肠杆菌—链霉菌穿梭质粒载体ｐＩＪ６５３ 中构建成表达载体ｐ ＷＹ１０１ 和ｐ ＷＹ１０２ ,用它们转化阿维菌素（ａｖｅｒｍｅｃｔｉｎｓ） 产生菌———阿维链霉菌（ Ｓｔｒｅｐｔｏｍｙｃｅｓ ａｖｅｒｍｉｔｉｌｉｓ） ,经Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ 分析并未检测到ｖｈｂ 基因表达,但用穿梭载体ｐＨＺ１２５２（ 其中的ｖｈｂ 基因位于受硫链丝菌素诱导的链霉菌强启动子ＰｔｉｐＡ之下） 转化阿维链霉菌并经硫链丝菌素诱导,则在该菌中表达出了有活性的ＶＨｂ 蛋白。ｐＨＺ１２５２ 在阿维链霉菌中发生了重组缺失,但缺失的ｐＨＺ１２５２ 上仍含有完整的ｖｈｂ 基因及诱导型强启动子,且可在阿维链霉菌中稳定遗传,却不能再转化大肠杆菌。     

Protease activity in cells ofBacillus megaterium during derepression

A proteolytic activity hydrolyzing denatured proteins of Bacillus megaterium labelled with 35S or 14C amino acids was detected in cells of the asporogenic strain of Bacillus megaterium. The substrate is hydrolyzed by the enzyme or enzymes at optimum pH around 7, their activity being almost completely inhibited by EDTA and o-phenanthroline. PMSF, the inhibitor of serine proteases, is slightly inhibitory. Gel filtration on a Sephadex column separated the protease activity to two or three fractions. The protease activity in cells with the repressed synthesis of protease corresponds to 5-20 mug of substrate degraded per hour by 1 mg of protein at 37 degrees C. It increases five to ten-fold during the derepression. When the intracellular protease activity increases the extracellular enzyme begins to be excreted into the medium. The intracellular protease activity rapidly decreases after the addition of chloramphenicol or of a mixture of amino acids to the derepressed culture. Half or even more of the protease activity is released from the cells during their conversion to protoplasts by means of lysozyme. This "periplasmic" activity remains mostly in the supernatant also after mesosomes have been centrifuged down from the periplasm. A portion of the activity bound in protoplasts sediments together with membrane fraction after their lysis.     

Characterization of Erwinia chrysanthemi extracellular proteases: cloning and expression of the protease genes in Escherichia coli.

Erwinia chrysanthemi, a phytopathogenic enterobacterium, secretes three antigenically and structurally distinct proteases, A, B, and C and produces a protease inhibitor, a low-molecular-weight, heat-stable protein which remains mostly intracellular and which binds specifically to the A, B, and C proteases. The structural genes for proteases A, B, and C and for the inhibitor are clustered on a ca. 40-kilobase DNA fragment present in cosmid pEW4. Escherichia coli strains harboring pEW4 secrete the three proteases into the medium during the exponential phase of growth, without intracellular accumulation and in the absence of detectable cell lysis. An 8.5-kilobase EcoRI fragment derived from the cosmid encodes proteases B and C and the inhibitor as well as functions involved in the synthesis or secretion (or both) of the proteases. The inhibitor is not required for protease synthesis or secretion.     

An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I

A method was developed to eliminate the proteases contaminating commercial DNase I, which can cause degradation of target protein during the purification process. Bio Basic DNase stock solution (in Tris–HCl buffer [pH 8.0] containing 5 mM CaCl₂) was first incubated at 50 °C to generate autolysis of proteases and zymogens, leading to a significant reduction in protease activity while preserving DNase activity. The residual protease activity was completely inhibited by further incubation with 2 mM PMSF (phenylmethylsulfonyl fluoride) or 2× S8830 inhibitor cocktail. This approach could be readily applicable to eliminate the protease activity in any DNase products or during the preparation of commercial DNase.     

Isolation and Characterization of a Proteinaceous Protease Inhibitor from Bacillus subtilis

A proteinaceous protease inhibitor which might have an intracellular role in modulating protease activity during sporulation was isolated from B. subtilis IFO 3027 by trichloroacetic acid and ethanol precipitations, and column chromatographies on SP-Sephadex, DEAE-Sephadex, DE AE-Toyopearl and Sephadex G-75.

The molecular weight of the inhibitor was estimated by gel filtration and SDS polyacrylamide gel electrophoresis to be about 16,000. The isoelectric point was determined as pH 4.8. The inhibitor is an acid and thermostable protein. The-amino acid sequence in the amino terminal region was determined to be (Met)-Glu-Asn-Gln-Glu-Val-Val-Leu-X-X-Asp-Ala-Ile-Gln-Glu- ··· (X, unidentified).

In addition to cytoplasmic serine proteases of the inhibitor-producing strain, the inhibitor inhibits various microbial serine proteases.     

Stabilization of proteolytic enzymes in solution

The stability of the neutral and alkaline proteases in a Bacillus subtilis enzyme mixture was studied in aqueous solutions at room temperature. Stabilization of the proteases in solution for periods up to 25 days was achieved by the addition of various protein preparations including casein and soya protein. The degree of stabilization by casein was concentration dependent to about 2% protein. The instability of the neutral protease in solutions of the B. subtilis enzyme mixture was shown to be due primarily to proteolysis by the alkaline protease since the diisopropylfluorophosphate-treated enzyme was quite stable. Formulation of such enzyme solutions at low pH gave greater stability as did solutions containing an alkaline protease inhibitor from potatoes. A Conceptual approach to the formulation of enzyme solutions containing proteolytic enzyme to ensure maximum stability is proposed.     

Evidence for a defective thiol protease inhibitor in skeletal muscle of mice with hereditary muscular dystrophy

The thiol protease inhibitor (TPI-d) from hind-limb skeletal muscle of dystrophic 60-day-old male mice (strain 129/ReJ/dy) has been purified to apparent homogeneity and compared with the thiol protease inhibitor (TPI-n) from hind-limb skeletal muscle of normal 60-day-old male littermates. While both TPI-d and TPI-n displayed identical properties on sodium dodecyl sulfate-polyacrylamide gels (14,800 relative mass), analytical isoelectric focusing gels (pI 4.5), and high performance liquid chromatography columns, TPI-d was unable to inhibit papain and cathepsin B after purification by isoelectric focusing. However, a component in the purified TPI-d preparation with an isoelectric point of 4.9 initially masked the functional state of TPI-d, using papain when assayed with the test proteases papain and cathepsins H and L. This inhibitory component was absent from TPI-n preparations. Pure TPI-d was also unable to inhibit in vitro myosin hydrolysis by cathepsin B, whereas TPI-n completely blocked cathepsin B catalyzed myosin hydrolysis. Given the central role of the thiol proteases, especially cathepsin B, in intracellular protein metabolism and the possibility that uncontrolled thiol protease activity in muscle leads to muscle protein breakdown and dystrophy, our data suggest that a modified (defective) thiol protease inhibitor (TPI-d) may be (one of) the end product(s) of the dystrophy gene in mice with the hereditary form of the disease.     

The 26S proteasome: ubiquitin-mediated proteolysis in the tunnel

The 26S proteasome is a self-compartmentalizing protease responsible for the degradation of intracellular proteins. This giant intracellular protease is formed by several subunits arranged into two 19S polar caps-where protein recognition and ATP-dependent unfolding occur-flanking a 20S central barrel-shaped structure with an inner proteolytic chamber. Proteins targeted to the 26S proteasome are conjugated with a polyubiquitin chain by an enzymatic cascade before delivery to the 26S proteasome for degradation into oligopeptides. As a self-compartmentalizing protease, the 26S proteasome circumvents proteins not destined for degradation and can be deployed to the cytoplasmic and nuclear compartments. The 26S proteasome is a representative of emerging group of giant proteases, including tricorn protease, multicorn protease, and TPPII (tripeptidyl peptidase II).     

Investigation of sequential behavior of carboxyl protease and cysteine protease activities in virus-infected Sf-9 insect cell culture by inhibition assay

Proteases produced during the culture of Spodoptera frugiperda Sf-9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV) were assayed with various protease inhibitors. This inhibitory analysis revealed that: (1) carboxyl and cysteine proteases were predominantly produced by the insect cells infected with recombinant AcNPV, the gene of which encoded a variant of green fluorescent protein in a portion of the polyhedrin gene of the baculovirus, and (2) the protease activity was almost completely blocked by pepstatin A (carboxyl protease inhibitor) and E64 (cysteine protease inhibitor) in an additive manner in the presence of EDTA. Utilizing the additive property of the inhibitors, the inhibition-based protease assay discriminated between the two protease activities and elucidated the sequential behavior of the carboxyl and cysteine proteases produced in the virus-infected Sf-9 cell culture. The carboxyl protease(s) existed in the virus-infected cells all the time and their level in the medium continuously increased. Uninfected cells also contained a carboxyl protease activity, the level of which was similar to that of the virus-infected cells. At a certain time after virus infection, the cysteine protease activity was largely increased in the virus-infected cells and a significant amount of the protease(s) was released into the medium, due to the cell membranes losing their integrity. The behavior of intracellular and extracellular cysteine protease activities coincided with that of a recombinant protein whose expression was under the control of the viral polyhedrin promoter. Similar examinations with wt-AcNPV-infected and uninfected insect cells showed that the inhibition-based protease assay was useful for analyzing the carboxyl protease and cysteine protease activities emerging in the insect cell (Sf-9)/baculovirus expression system.     

Sequential activation of caspases and serine proteases (serpases) during apoptosis

Analogous to caspases, serine (Ser) proteases are involved in protein degradation during apoptosis. It is unknown, however, whether Ser proteases are activated concurrently, sequentially, or as an alternative to the activation of caspases. Using fluorescent inhibitors of caspases (FLICA) and Ser proteases (FLISP), novel methods to detect activation of these enzymes in apoptotic cells, we demonstrate that two types of Ser protease sites become accessible to these inhibitors during apoptosis of HL-60 cells. The prior exposure to caspases inhibitor Z-VAD-FMK markedly diminished activation of both Ser protease sites. However, the unlabeled inhibitor of Ser-proteases TPCK had modest suppressive effect- while TICK had no effect- on the activation of caspases. Activation of caspases, thus, appears to be an upstream event and likely a prerequisite for activation of FLISP-reactive sites. Differential labeling with the red fluorescing sulforhodamine-tagged VAD-FMK and the green fluorescing FLISP allowed us to discriminate, within the same cell, between activation of caspases and Ser protease sites. Despite a certain degree of co-localization, the pattern of intracellular caspase- vs FLISP- reactive sites, was different. Also different were relative proportions of activated caspases vs Ser protease sites in individual cells. The observed induction of FLISP-binding sites we interpret as revealing activation of at least two different apoptotic Ser proteases; by analogy to caspases we denote them serpases. Their apparent molecular weight (62-65 kD) suggests that they are novel enzymes.