柱状田头菇（茶薪菇）金属硫蛋白的分离纯化与特性研究

应用快速灌注色谱系统首次从经Cd^2 诱导的柱状田头菇(茶薪菇)Agrocybe Cylindracea(DC.:Fr:)R.Maoire菌丝体中分离得到一种镉结合蛋白。通过Sephadex G-75凝胶过滤层析,原子吸收光谱分析(AAS),巯基含量测定及紫外吸收光谱分析表明这种镉结合蛋白具有金属硫蛋白(metallothionein,MT)的理化性质：即分子量为6kDa、每分子MT含18个半胱氨酸残基并结合7个镉原子、具有镉硫金属簇的特征紫外吸收光谱,初步鉴定为茶薪菇Cd—MT。     

柱状田头菇(茶薪菇)金属硫蛋白的分离纯化与特性研究*

应用快速灌注色谱系统首次从经Cd2+诱导的柱状田头菇(茶薪菇)Agrocybecylindracea(DC.:Fr:)R.Maoire菌丝体中分离得到一种镉结合蛋白。通过SephadexG-75凝胶过滤层析,原子吸收光谱分析(AAS),巯基含量测定及紫外吸收光谱分析表明这种镉结合蛋白具有金属硫蛋白(metallothionein,MT)的理化性质:即分子量为6kDa、每分子MT含18个半胱氨酸残基并结合7个镉原子、具有镉硫金属簇的特征紫外吸收光谱,初步鉴定为茶薪菇Cd-MT。     

鼠肝中金属硫蛋白的纯化

旨在探索鼠肝中金属硫蛋白(MT)提取工艺并加以改进。按照经典方法工艺提取MT,用中空纤维柱超滤方法加以改进,依据MT自身特点进行分离和鉴定。结果显示,粗品符合MT特点:分子量在6.5kD左右;无280nm特征吸收峰,加酸后紫外吸收(200-300nm)肩峰消失;通过离子交换可以实现MT1、MT2的分离;通过原子吸收测定,含有锌、铜、镉3种金属,锌含量最高,具有重要的病理生理意义。因此,与其它方法比较,改进后方法更适宜实验室制备。     

高耐镉膨胀肾形虫种群金属硫蛋白基因的鉴定与诱导表达

为研究金属硫蛋白(Metallothionein, MT)对生物体抵抗重金属毒性的效应,获得一种具有镉高耐受性的膨胀肾形虫(Colpoda inflata)东北种群,该种群96h最高镉耐受浓度10 mg/L,其金属硫蛋白含量表现出与镉浓度、肾形虫种群增长率存在正相关关系。克隆获得金属硫蛋白Col-MT1基因,对基因序列和氨基酸序列特征分析表明,其为金属硫蛋白基因家族7a亚型的新成员。qRT-PCR实验证实, Col-MT1基因在60h、84h和108h三个时间点对5种浓度镉胁迫均上调表达,与镉浓度之间呈现出一定的剂量-效应关系。其分子调控机制还有待进一步研究。上述结果补充了原生动物MT基因数据库,为进一步揭示C. inflata MT基因的功能,以及应用于镉污染监测和环境修复奠定了基础。     

镉诱导金属硫蛋白在华溪蟹组织中的表达

采用体外暴露染毒法,研究了不同浓度与时间条件下,镉诱导河南华溪蟹(Sinopotamon henanense)金属硫蛋白(metallothionein,MT)在肝胰腺、肌肉、鳃和卵巢中的表达差异。镉浓度分别为0、14·5mg/L、29mg/L和58mg/L;处理时间依次为1d、3d和5d。利用镉血红蛋白饱和法和火焰原子吸收分光光度法(AAS)测定MT的蛋白含量。结果显示,用不同的染毒浓度和处理时间,镉在组织中诱导产生MT的含量有较大差异,其中肝胰腺MT的诱导量最大,变化规律也最明显;肌肉中也有较大量MT的表达;而鳃和卵巢MT的诱导量均较低。此外,本文分析了镉的浓度与时间梯度对诱导MT表达的影响与毒性效应机制。结论:组织不同,染毒浓度及时间不同,镉诱导MT的表达也不同,具有一定的组织差异性和规律性。     

灌注色谱法快速分离纯化茶薪菇金属硫蛋白

为建立一种快速高效分离Cd诱导茶薪菇金属硫蛋白（MT）的方法,以灌注色谱技术为平台,采用BioCAD 700E灌注色谱系统分离纯化Cd诱导茶薪菇金属硫蛋白,并对色谱条件进行了优化,结果表明：经优化相关参数（缓冲液pH值8．2,流速5ml/min,洗脱体积10CV）建立的快速灌注色谱法能高效地进行茶薪菇Cd-MT分离,且具有快速、高效、自动化操作、易放大等优点。     

镉暴露大鼠睾丸支持细胞金属硫蛋白表达的时相研究

啮齿目动物的睾丸较肝脏对镉毒性更为敏感.为阐明不同组织细胞的镉毒性分子机制,对镉暴露大鼠睾丸支持细胞与肝脏金属硫蛋白基因(MT)的表达及镉蓄积进行了时相研究.成年雄性SD大鼠低剂量镉(4.0 μmol/kg)皮下注射后,立即进行睾丸支持细胞和肝脏组织的分离.采用RT-PCR技术分析mRNA,并用光密度扫描作半定量分析,蛋白质定量用ELISA方法,原子分光光度吸收法测定镉浓度.结果显示：镉暴露1 h后肝脏MT mRNA即有明显的诱导表达,3 h达高峰;支持细胞也有明显的诱导表达, 6 h达高峰.镉暴露后肝脏MT有明显的诱导表达,但睾丸支持细胞不但未见MT增加而且还稍有下降.提示：a.镉对MTmRNA的诱导表达具有时间依赖性和组织特异性.b.镉虽然能诱导睾丸MT的转录,但没有促进其MT的合成,这可能是睾丸对镉毒性与致癌作用较肝脏更敏感的重要原因.     

镉致黑斑蛙肝脏氧化损伤与金属硫蛋白含量的变化

为观察镉对黑斑蛙(Rana nigromaculata)肝脏脂质过氧化产物和金属硫蛋白含量的影响,将黑斑蛙暴露于10.0mg·mL-1浓度的镉溶液中30d,分别测定了黑斑蛙在暴露4、10d和30d时肝脏组织中镉(Cd)、还原型谷胱甘肽(GSH)、金属硫蛋白(MT)和过氧化产物丙二醛(MDA)的含量。实验结果表明,黑斑蛙肝脏中镉的积累量、GSH和MT含量均随着镉暴露时间的延长而显著升高,具有明显的时间-效应关系;在镉暴露的第10天,肝MDA含量明显高于对照组。提示镉可对黑斑蛙肝脏造成氧化损伤,而GSH、MT含量的升高则可能是机体抗氧化损伤的机理之一。     

力竭游泳诱导的大鼠睾丸金属结合蛋白的初步分离与鉴定

目的初步分离和鉴定力竭运动诱导的大鼠睾丸金属结合蛋白(testis metal-binding proteins,TMB-Ps)。方法 8只雄性SD大鼠一次性力竭游泳运动(8只对照)后6 h取睾丸和肝脏组织,用镉饱和法测定TMBPs和肝脏金属硫蛋白(metallothionein,MT)含量,用tricine-SDS-PAGE方法分离镉饱和法提取液的TMBPs组分和肝脏MT,用液相色谱-串联质谱法(LC-MS-MS)鉴定TMBPs分离条带。结果力竭运动组大鼠TMBPs水平(113.71±11.72)nmol Cd/g testis显著高于安静对照组(87.14±12.72)nmol Cd/g testis(P0.01),肝脏MT表达量(64.70±14.89)μg/g也显著高于安静对照组(7.32±3.31)μg/g(P0.001)。LC-MS-MS对tricine-SDS-PAGE分离的蛋白条带的鉴定结果为:TMBPs含有泛素、铜-锌-超氧化物歧化酶、酪蛋白样磷蛋白等蛋白质,另外还应包括MT。结论TMBPs由一组具有金属结合性、耐热性和诱导性的蛋白质所组成,主要有泛素、超氧化物歧化酶和酪蛋白样磷蛋白。镉饱和法并非测定金属硫蛋白的特异性方法。     

异常汉逊酵母BD102金属硫蛋白的分离纯化和鉴定

从异常汉逊酵母中分离出拮抗Cu²⁺、Cd²⁺等重金属、并经铜、镉诱导产生金属硫蛋白的异常汉逊酵母 (Hansenulaanomala)BD1 0 2。无细胞抽提液经SephadexG 50、DEAESepharoseCL 6B、SephadexG 2 5三次凝胶及阴离子交换柱层析分离纯化 ,Cu²⁺诱导得到Cu MTs两个亚型 ,Cd²⁺诱导得到Cd MT一个亚型。Mr分别约为 7kD和 7 5kD ,由 60和 61个氨基酸组成 ,其中半胱氨酸含量各为 6.8%和 1.0 %。每分子金属硫蛋白 (Cu MTs或Cd MT)可结合 4个铜或镉原子     

Purification and characterization of a biliverdin-associated protein from the hemolymph of Manduca sexta

A biliverdin binding protein, insecticyanin, has been isolated from the hemolymph of the fourth instar tobacco hornworm Manduca sexta. The protein has been purified to apparent homogeneity by conventional chromatography with a cumulative yield of 40-50%. The protein (Mw 71 600) is composed of three subunits (Mr 23 000). Each subunit binds one biliverdin molecule. Proton magnetic resonance spectroscopy and absorption spectroscopy demonstrate that the bilin is the biliverdin IX gamma isomer.     

连续流层析技术在亲和层析中的应用及生产放大评估

生物制药行业迅速发展,尤其是上游表达量的增加和规模的扩大,促使上游培养采用连续灌流方式,同时也推动了下游纯化生产工艺相应的采取连续纯化策略。以灌流培养的Fc融合蛋白为例,采用 BioSMB PD设备,对比了下游工艺亲和层析捕获步骤中单柱批次纯化和连续流层析纯化的样品纯度和收率,并在此基础上进行小试工艺放大和生产实际用量成本计算评估。连续流层析实现了上游灌流培养与下游亲和层析连续化的可行性,工艺稳定,回收率与批次纯化接近,但相比批次纯化,生产效率明显提高,填料载量提高,同时填料使用效率提高,生产成本显著降低。     

Gamma irradiating chromatography columns enables bioburden-free integrated continuous biomanufacturing

An important consideration for integrated continuous biomanufacturing is that the downstream chromatography steps integrated with the bioreactor should maintain a low bioburden state throughout the entire duration of the operation. One potential strategy to achieve this is to start bioburden-free and functionally close the chromatography system. While chromatography skids themselves can be rendered bioburden-free, limitations exist in applying these methods to chromatography columns. The small column sizes used in continuous multicolumn chromatography enable gamma irradiation of disposable columns to render them bioburden-free. However, this approach has not been widely implemented, likely because gamma irradiation can negatively impact resin performance. Here, several protective mobile-phase modifiers were screened and shown to help chromatography resins retain naïve-like performance. Gamma irradiated columns were then integrated into perfusion bioreactors for continuous capture. Successful integrated continuous capture downstream of perfusion bioreactors for greater than 40 days using protein A, custom affinity, and non-affinity capture resins for multiple biologic modalities is demonstrated in development and commercial settings. No indications of time-based performance decline or bioburden growth have been observed. This strategy enables bioburden-free integrated continuous biomanufacturing operations and could allow full process closure and decreased environmental control requirements for facilities; thus, permitting simultaneous multi-product operations in a ballroom arrangement.     

Purification and characterization of trichomaglin--a novel ribosome-inactivating protein with abortifacient activity

Trichomaglin, a novel ribosome-inactivating protein, has been isolated from root tuber of a plant Maganlin (Trichosanthes Lepiniate, Cucurbitaceae). The isolation and purification procedure included ammonium sulfate precipitation, Sephadex G-75 chromatography and CM-Sephadex C-50 chromatography. The protein was identified to be homogeneous by SDS-PAGE and FPLC analysis. Its molecular weight is 24,673 dalton and isoelectric point is 5.8, determined by electrospray ionization mass spectroscopy and isoelectric focusing gel electrophoresis respectively. Trichomaglin can inhibit protein synthesis in rabbit reticulocyte lysate with ID50 of 10.1 nM. When rat ribosome was incubated with trichomaglin, a diagnostic RNA fragment appeared on polyacrylamide gel after ribosomal RNAs were treated with acidic aniline. It was concluded that trichomaglin is an RNA N-glycosidase. In addition, it has been verified to be an abortifacient protein.     

The disaggregation of calcium-depleted casein micelles

The effect of depletion of Ca2+ on the composition and size distribution of casein micelles in milk has been examined using chemical analysis, size exclusion chromatography, fast protein liquid chromatography, turbidimetry and photon correlation spectroscopy. Partial removal of Ca2+ by EDTA and subsequent dialysis resulted in disaggregation of some of the casein micelles; as the EDTA concentration increased, the proportions of Ca2+ and phosphate relative to protein in the micelles remaining intact decreased. However, the composition of the intact micelles, with respect to the different caseins, and the number-frequency size distribution were essentially unchanged.     

Expression, purification, and physicochemical characterization of the N-terminal active site of human angiotensin-I converting enzyme.

We have cloned, over expressed, and purified one of the two catalytic domains (residues Ala361 to Gly468, ACE-N) of human somatic angiotensin-I converting enzyme in Escherichia coli. This construct represents the N-catalytic domain including the two binding motifs and the 23 amino acid spacers as well as some amino acid residues before and after the motifs that might help in correct conformation. The overexpressed protein was exclusively localized to insoluble inclusion bodies. Inclusion bodies were solubilized in an 8-M urea buffer. Purification was carried out by differential centrifugation and gel filtration chromatography under denaturing conditions. About 12 mg of ACE-N peptide per liter of bacterial culture was obtained. The integrity of recombinant protein domain was confirmed by ESI/MS. Structural analysis using CD spectroscopy has shown that, in the presence of TFE, the ACE-N protein fragment has taken a conformation, which is consistent with the one found in testis ACE by X-ray crystallography. This purification procedure enables the production of an isotopically labeled protein fragment for structural studying in solution by NMR spectroscopy.     

Ergosterol is the only sterol in Kluyveromyces fragilis

The sterol fraction has been extracted from cells of Kluyveromyces fragilis and analyzed by thin-layer chromatography, UV spectroscopy, gas-liquid chromatography and mass spectroscopy. Only ergosterol could be detected.