亲和介质及溶液条件对蛋白质溶液中内毒素去除的影响

生物制品中内毒素的去除是一项十分重要的工作。为了更好地去除各种生物制品中的内毒素,采用合成的多粘菌素B琼脂糖亲和介质,通过静态吸附的方法去除蛋白质溶液中的内毒素。重点考察了介质的间臂长度、配基密度以及各种溶液条件(pH值、盐种类和浓度、蛋白质种类和浓度、内毒素浓度、添加剂等)对内毒素去除率及蛋白质回收率的影响。分别采用动态浊度法和Lowry法检测内毒素含量和蛋白质浓度。结果表明该介质具有载量高、去除速度快、去除率高、可重复使用的特点。此外,配基密度、pH值、盐浓度和蛋白质特性(等电点和疏水性)对内毒素去除效果均有重要影响。在优化的条件下,血红蛋白、人血清白蛋白和溶菌酶的回收率分别达到87.2%、73.4%和97.3%,相应的内毒素去除率分别达到99.8%、97.9%和99.7%。阐明了各种因素对内毒素去除率和蛋白质回收率的影响规律,为生物制品中内毒素的高效去除提供了参考。     

响应面法建立层析纯化去除百日咳丝状血凝素中内毒素工艺

【背景】无细胞组分百日咳疫苗在人群中接种后不良反应发生率大大降低,是未来百日咳疫苗的发展方向,但是新的抗原纯化方式需要工艺中加入内毒素的去除。【目的】使用响应面法优化层析纯化法去除无细胞百日咳疫苗中百日咳丝状血凝素(Filamentous hemagglutinin,FHA)中内毒素的工艺。【方法】通过单因素试验,确定响应面设计范围;根据响应面法设计原理,使用Mini TAB软件,以FHA的回收率和收获的FHA蛋白浓度,同时兼内毒素合格为考察指标,对上样样品量、样品p H、样品电导Cond进行优化,最终确定去除FHA内毒素的层析纯化工艺。【结果】使用目前的层析纯化条件获得的Capto adhere去除FHA的内毒素的最佳工艺条件:p H 5.3,Cond 9.6,Mass 3.0。【结论】用响应面法优化了去除百日咳丝状血凝素中内毒素的层析纯化工艺,这种方法效率高、耗时少,为后续生物制品工艺扩大再生产提供参考。     

单克隆抗体纯化过程中内毒素去除方法分析

目的：分析单克隆抗体纯化过程中,去除内毒素的不同方法的应用效果,并探讨它们应用于中试规模的可行性。方法与结果：比较了多聚赖氨酸型的内毒素去除填料、20nm膜过滤、将单抗附着在蛋白A柱上后使用精氨酸和组氨酸溶液冲洗等3种方法的内毒素去除效果,发现3种方法都可以将内毒素水平大幅降低,可分别将内毒素去除70％、88％和97％。因为单抗分子等电点较高,所获得的最低内毒素含量为0．2～0-3EU／mg。结论：3种方法均具有一定的工艺放大潜力,进一步提高内毒素去除效果将需要综合使用不同机理的去除技术。     

一种简便易行核酸疫苗质粒纯化工艺的开发

介绍了一种成本低、步骤少、简单易行的质粒纯化制检工艺。该工艺选择优势产生超螺旋质粒的大肠杆菌菌株以无蛋白质培养基进行发酵罐培养,采用碱裂解法,对质粒制备过程中所用的层析吸附材料、核酸结合溶液、去除内毒素等杂质的方法和浓缩等步骤进行了实用性改进,并建立了相应的检定方法,所得质粒的纯度达到临床级要求。     

碱裂解法提取质粒DNA的研究

对质粒DNA及其结构、形态和功能进行了综述 ,介绍了碱裂解法提取质粒DNA的基本步骤 ,并对其提取过程中的注意事项进行了剖析 ,分析比较了不同提取方法的优点     

水稻内生链霉菌中的线型和环型质粒

植物内生放线菌的研究是一个近年来兴起的学科领域,在进一步探索和开发微生物资源方面,植物内生放线菌逐渐成为相关领域同行的关注热点.     

内毒素法兔发热动物模型及其标准化的研究

目的探讨不同剂量的大肠埃希菌内毒素(LPS),实验室的温湿度、动物的固定方法和动物的生理机能状态对兔发热反应的影响.方法实验在能控制温湿度的实验室内进行,静脉注射不同剂量(0.5?ng/kg\,2?ng/kg\,20?ng/kg\,100?ng/kg\,200?ng/kg\,2000?ng/kg)大肠埃希菌内毒素(Lipopolysaccharide,LPS)诱发兔发热反应.结果随LPS注射剂量的增加兔发热反应增强.表现在发热潜伏期逐渐缩短;发热高峰值逐渐增加;发热时程逐渐延长;体温反应指数逐渐加大.另外LPS注射剂量不同,动物发热曲线的峰型亦不相同,小剂量LPS(0.5?ng/kg及2?ng/kg)可诱发单峰型发热反应;用量增加到20?ng/kg以上时,呈双峰型发热反应.静脉注射剂量为100～200?ng/kg时兔发热反应呈典型的双峰热,且发热反应均一,是造模的适宜剂量.实验结果证明实验室的温度为25℃左右动物以颈部固定方法为宜.     

费氏中华根瘤菌内源质粒的不相容性及其在质粒消除中的应用

用Tn5 Mob sacB转座子标记费氏中华根瘤菌 (Sinorhizobiumfredii)HN0 1和WWG1 8的内源质粒 ;在含有 1 0 %蔗糖的TY培养基上进行了质粒消除试验以鉴定其功能。将HN0 1的标记共生质粒pSymHN0 1b转入费氏中华根瘤菌WWG1 8SR和C361SR中 ,质粒快速检测、质粒消除和植物盆栽结瘤试验结果证明 :HN0 1的共生质粒与WWG1 8SR的共生质粒具有不相容性 ,但能与其非共生质粒相容。相反 ,HN0 1的共生质粒可与C361SR的共生质粒相容 ,但与其中一个非共生质粒具有不相容性。利用费氏中华根瘤菌不同菌株质粒间的不相容性 ,本研究成功地消除了WWG1 8SR的共生质粒和C361SR的一个非共生质粒。     

抗鞘翅目δ－内毒素 及毒素基因文库的构建

研究了对鞘翅目昆虫有毒效的5个苏云金芽孢杆菌新菌株YM-03、Sph04-04、YK14-01、SH11-05、Sph16-01伴孢晶体的蛋白质组成,以柳蓝叶甲为供试虫测定了它们的LC_(50)值,其中YM-03毒力最高。测定了YM-03晶体蛋白N-末端部分氨基酸序列。用琼脂糖凝胶电泳快速检测了5株菌的质粒组成,证明其质粒图型各不相同。以广谱的cosmid质粒pLAFRI为载体,通过限制酶EcoRI部分酶解获得“目的”DNA片段,构建了菌株YM-03的总DNA文库,对17个抗性克隆的质粒进行酶切分析表明,其中含有外源片段的克隆占总数的76％,超过要求的理论值。以人工合成的杀鞘翅目基因的18bp保守序列片段为探针,筛选了近1200个抗性克隆,获得了3个阳性克隆,LE392(PBYM2)、LE392(pBYM3)和LE392(pBYM4),毒力测定试验表明LE392(pBYM3)和LE392(pBYM4)有一定表达,表明其携带有δ-内毒素基因。     

Triton X-114-aided purification of latent tyrosinase

Mushroom tyrosinase was partially purified using an aqueous two-phase system with Triton X-114. The purification achieved was 5.5-fold from a crude extract of mushroom pileus, with a high recovery of 84%. The phenols were reduced to 8% of the original content, avoiding pre- and post-purification tanning of the enzyme. The enzyme obtained was latent and was activated 3-fold by trypsin, 2.7-fold by changes in the pH and to different extents by cationic and anionic detergents, the latter being the more effective. There was also a synergistic effect between trypsin and detergent, at low detergent concentrations. When kinetically characterized, latent enzyme showed both monophenolase and diphenolase activities, the latter activity displaying an unexpected lag period before reaching the steady-state rate. This behaviour is characteristic of a hysteretic enzyme, and has not been previously described for this enzyme. In addition, inhibition studies with substrate analogues were carried out, tropolone being found to be the most effective inhibitor.     

Ciliary membrane tubulin and associated proteins: a complex stable to Triton X-114 dissociation

When either membranes from scallop gill cilia or reconstituted membranes from the same source are solubilized with Triton X-114 and the detergent is condensed by warming, no significant fraction of any major membrane protein partitions into the micellar detergent. Rather, most of the membrane lipids condense with the detergent phase, forming mixed micelles from which nearly pure lipid vesicles may be produced by adsorption of detergent with polystyrene beads. One minor membrane protein, with a molecular weight of about 20000, is associated consistently with these vesicles. The aqueous phase contains a fairly homogeneous protein-Triton X-114 micelle sedimenting at 2.6 S in the analytical ultracentrifuge. Sucrose gradient velocity analysis in a detergent-free gradient indicates moderate size polydispersity but constant polypeptide composition throughout the sedimenting protein zone. Sucrose gradient equilibrium analysis (also in a detergent-free gradient) results in a protein-detergent complex banding at a density of 1.245 g/cm³. Sedimentation of the protein-detergent complex in the ultracentrifuge, followed by fixation and normal processing for electron microscopy, reveals a fine, reticular material consisting of 5–10-nm granules. These data are consistent with previous evidence that membrane tubulin and most other membrane proteins exist together as a discrete lipid-protein complex in molluscan gill ciliary membranes.     

Hydrophobicity of protochlorophyllide oxidoreductase, characterized by means of Triton X-114 partitioning of isolated etioplast membrane fractions

The inner etioplast membrane possesses a pronounced lateral heterogeneity with respect to protein and lipid composition as well as ultrastructural appearance. Little is known about the reason for formation of the regular branched structure shown by the prolamellar body part of the membrane. A specific interaction between the membrane lipids and the dominating protein NADPH-protochlorophyllide oxidoreductase (PCR, EC 1.6.99.1) might be of major importance. In this study isolated prolamellar bodies and prothylakoids from the leaves of dark-grown wheat ( Triticum aestivum L. cv. Starke II, Weibull) were exposed to Triton X-114 partitioning in media with 150 m M NaCl and without. By comparing the partitioning of PCR, the ATP synthase (EC 3.6.1.3) polypeptides, and ribulosebisphosphate carboxylaseoxygenase (EC 4.1.1.39) in the different systems, it was concluded that PCR is an integral membrane protein with substantial hydrophilic domains.     

Release of remnant plasma membrane from milk fat globules by Triton X-100

The nonionic detergent, Triton X-100, was investigated as an agent for releasing plasma membrane from milk fat globules. The sedimentable material ($\text{50 000 × g}$, 1 h) derived by treating washed goat globules with the detergent (0.2%) was compared to membrane made by the classical globule churning procedure. Characterization included lipid and protein analyses, gel electrophoresis of peptide components, determination of enzymatic activities, and examination with the electron microscope. The results established that the detergent-released material is membrane with similarities to the product by churning. Evaluation of variables revealed that a detergent concentration of 0.1 to 0.2% and reaction temperature of 20–22°C appear optimum with respect to membrane yield when a reaction time of 2 min is employed. At higher detergent concentrations or temperatures removal of phospholipid from the membrane was maximized. Triton X-100 was observed to release membrane from milk fat globules of the goat, human and cow, the latter with a minor procedural modification. The detergent based method is a convenient procedure for obtaining plasma membrane material in good yield for biochemical studies. It also should aid investigations of milk fat globule structure.     

The effect of n-butanol on Triton X-114 phase partitioning

n-Butanol interferes with the fractionation of amphiphilic and hydrophilic molecules during the Triton X-114 phase separation procedure. The indicators oil red (hydrophobic) and p-nitrophenol (hydrophilic) were useful for predicting the effectiveness of the Triton X-114 partition method. For n-butanol extracts containing oil red, 5-nucleotidase, or alkaline phosphatase, the hydrophobic molecules and Triton X-114 were retained in the aqueous phase during incubations at 30°C. The n-butanol interference was concentration-dependent and was reduced by lowering the final n-butanol concentration of the sample to 1.5% (v/v) or less. The results demonstrate how buffer-diluted n-butanol extracts of 5-nucleotidase and alkaline phosphatase can be successfully employed for subsequent Triton X-114 fractionation of the enzymes.This revised version was published online in October 2005 with corrections to the Cover Date.     

Identification and characterization of a Triton X-100 replacement for virus inactivation

Triton X-100 detergent treatment is a robust enveloped virus inactivation unit operation included in biopharmaceutical manufacturing processes. However, the European Commission officially placed Triton X-100 on the Annex XIV authorization list in 2017 because a degradation product of Triton X-100, 4-(1,1,3,3-tetramethylbutyl) phenol (also known as 4-tert-octylphenol), is considered to have harmful endocrine disrupting activities. As a result, the use of Triton X-100 in the European Economic Area (EEA) would not be allowed unless an ECHA issued authorization was granted after the sunset date of January 4, 2021. This has prompted biopharmaceutical manufacturers to search for novel, environment-friendly alternative detergents for enveloped virus inactivation. In this study, we report the identification of such a novel detergent, Simulsol SL 11W. Simulsol SL 11W is an undecyl glycoside surfactant produced from glucose and C11 fatty alcohol. We report here that Simulsol SL 11W was able to effectively inactive enveloped viruses, such as xenotropic murine leukemia virus (XMuLV) and pseudorabies virus (PRV). By using XMuLV as a representative enveloped virus, the influence of various parameters on the effectiveness of virus inactivation was evaluated. Virus inactivation by Simulsol SL 11W was effective across different clarified bioreactor harvests at broad concentrations, pH, and temperature ranges. Simulsol SL 11W concentration, temperature of inactivation, and treatment time were identified as critical process parameters for virus inactivation. Removal of Simulsol SL 11W was readily achieved by Protein A chromatography and product quality was not affected by detergent treatment. Taken together, these results have shown the potential of Simulsol SL 11W as a desirable alternative to Triton X-100 for enveloped virus inactivation that could be readily implemented into biopharmaceutical manufacturing processes.     

Spectroscopic properties of protochlorophyll forms in Triton X-100 detergent micelles

Protochlorophyll (PChl) forms were performed in Triton X-100 detergent micelles. The concentration of Triton X-100 was 7·10^?4 M (above the critical micellar concentration); the concentration of PChl varied between 1.6·10^?5 and 1.8·10^?4 M. Absorption, fluorescence and circular dichroism (CD) spectra were registered. The absorption spectra were resolved into Gaussian components by computer analysis. PChl forms with absorption bands at 632–634, 638, 652–654, 663–664, 668 and 676 nm and with fluorescence emission bands at 634–636, 640–644, 652–655, 677–678, 686 and 694–696 nm were observed in micellar solutions of different PChl concentrations. The CD spectra showed a strong dependence on the concentration of PChl: positive CD signals or positive Cotton effects were observed in the vicinity of 650 nm. The intensity of these signals increased in parallel with increasing concentration of PChl. No CD signals were found in the region of the longer wavelength absorption bands. These data show that the PChl exists in many different forms in this system, and the spectroscopic properties of these forms are determined by different molecular interactions viz., interactions of PChl with Triton X-100 or water molecules and/or by the aggregation of PChl.     

The effect of cholesterol on the solubilization of phosphatidylcholine bilayers by the non-ionic surfactant Triton X-100

Most of the studies on the solubilization of model membranes by Triton X-100 (TR) involve one lipid. The aim of the present study was to evaluate the effect of the addition of cholesterol on the solubilization of bilayers made of palmitoyloleoylphosphatidylcholine (POPC) or dipalmitoylphosphatidylcholine (DPPC). Detailed investigation of the kinetics of solubilization of the cholesterol-containing bilayers by TR at different temperatures reveals that: (i) At 4 degrees C, solubilization of both systems is relatively slow. Hence, in order to prevent misleading conclusions from turbidity measurements it is important to monitor the solubilization after steady-state values of optical density (OD) are reached. (ii) Studies of the temperature-induced changes of the aggregates present in mixtures of TR, POPC and cholesterol indicate that the state of aggregation at all temperatures (including 4 degrees C) represents equilibrium. By contrast, for DPPC/cholesterol/TR mixtures "kinetic traps" may occur not only at 4 degrees C but at higher temperatures as well (e.g. 37 degrees C). (iii) The presence of cholesterol in POPC bilayers makes the bilayers more resistant to solubilization at low temperatures, especially at 4 degrees C. As a consequence, the temperature dependence of the TR concentration required for complete solubilization (Dt(sol)) is no longer a monotonically increasing function (as for POPC bilayers) but a bell-shaped function, with a minimum at about 25 degrees C. Inclusion of cholesterol in DPPC bilayers makes the bilayers more resistant to solubilization at all temperatures except 4 degrees C. In this system, we observe a bell-shaped dependence of Dt(sol) on temperature, with a minimum at 37 degrees C. (iv) Both the rate of vesicle size growth and the rate of the solubilization of POPC vesicles are not affected by the inclusion of cholesterol in the bilayers. Similarly, cholesterol did not affect significantly the rate of size growth of DPPC bilayers at all temperatures, but reduced the rate of solubilization at 4 degrees C.     

Tandem application of cationic colloidal silica and Triton X-114 for plasma membrane protein isolation and purification: towards developing an MDCK protein database

Plasma membrane (PM) proteins are attractive therapeutic targets because of their accessibility to drugs. Although genes encoding PM proteins represent 20-30% of eukaryotic genomes, a detailed characterisation of their encoded proteins is underrepresented, due, to their low copy number and the inherent difficulties in their isolation and purification as a consequence of their high hydrophobicity. We describe here a strategy that combines two orthogonal methods to isolate and purify PM proteins from Madin Darby canine kidney (MDCK) cells. In this two-step method, we first used cationic colloidal silica (CCS) to isolate adherent (Ad) and non-adherent (nAd) PM fractions, and then subjected each fraction to Triton X-114 (TX-114) phase partitioning to further enrich for hydrophobic proteins. While CCS alone identified 255/757 (34%) membrane proteins, CCS/TX-114 in combination yielded 453/745 (61%). Strikingly, of those proteins unique to CCS/TX-114, 277/393 (70%) had membrane annotation. Further characterisation of the CCS/TX-114 data set using Uniprot and transmembrane hidden Markov model revealed that 306/745 (41%) contained one or more transmembrane domains (TMDs), including proteins with 25 and 17 TMDs. Of the remaining proteins in the data set, 69/439 (16%) are known to contain lipid modifications. Of all membrane proteins identified, 93 had PM origin, including proteins that mediate cell adhesion, modulate transmembrane ion transport, and cell-cell communication. These studies reveal that the application of CCS to first isolate Ad and nAd PM fractions, followed by their detergent-phase TX-114 partitioning, to be a powerful method to isolate low-abundance PM proteins, and a useful adjunct for in-depth cell surface proteome analyses.     

Triton X—100加KI对燕麦根细胞质膜ATP酶的溶解

在酸性条件下,1％ Triton X—100加 0.25mol/L KI能有效地溶解燕麦根细胞质膜ATP酶。溶解的ATP酶水解ATP的最适pH在6.5左右,酶活性受到Na_3VO_4和DES的强烈抑制,而不受Na_2MoO_4和NaN_3的抑制。溶解的酶液经透析后,K~ —ATP酶活性占Mg~(2 ),KCl—ATP酶活性的85％。