The silica-binding Si-tag functions as an affinity tag even under denaturing conditions

We recently reported a one-step affinity purification method using a silica-binding protein, designated Si-tag, as a fusion partner and silica particles as the specific adsorbents (Ikeda et al., Protein Expr. Purif. 71 [2010] 91-95) [13]. In this study, we demonstrate that the Si-tag also binds to the silica surface even under denaturing conditions, thereby facilitating affinity purification of recombinant proteins from inclusion bodies. A fusion protein of the Si-tag and a biotin acceptor peptide (AviTag), which was expressed as inclusion bodies in Escherichia coli, was used as a model protein. To simplify our purification method, we disrupted recombinant E. coli cells by sonication in the presence of 8M urea with concomitant solubilization of the inclusion bodies. The fusion protein was recovered with a purity of 90 ± 3% and yield of 92 ± 6% from the cleared cell lysate. We also discuss the binding mechanism of the Si-tag to a silica surface in the presence of high concentrations of denaturant. We propose that the intrinsic disorder of the polycationic Si-tag polypeptide plays an important role in its binding to the silica surface under denaturing conditions.     

Use of epitope tags for routine analysis of transgene expression

Peptide and RNA epitope tags as tools for routine analysis of transgene expression and protein accumulation in transformed plant cell cultures was evaluated using three genes that encode very structurally and functionally different proteins. A T7 peptide was introduced at the amino- and carboxyl-termini of phosphinothricin-N-acetyl transferase and avidin and at the carboxyl-terminus of galactose oxidase. An RNA sequence that forms a higher order structure that is recognized by antibodies raised against the FLAG peptide was separately introduced into the 3 nontranslated region of these genes. Constructs were introduced into maize cell cultures using particle bombardment and transgene expression, protein accumulation, protein function and presence of the tags in RNA and/or protein as appropriate were evaluated in up to approximately 25 culture lines per construct. Results indicate that, while there will likely always be a need for some empirical evaluation of any tag-protein combination, introduction of the peptide tag at the amino-terminus was generally more successful than was incorporation at the carboxyl-terminus. RNA tags show promise for this purpose, but routine application will require development of a very sensitive immunoassay.Both of these authors contributed equally to this work and should be recognized as first authors.     

Initial conformational changes of human transthyretin under partially denaturing conditions

Human transthyretin (TTR) is an amyloidogenic protein. The pathway of TTR amyloid formation has been proposed based on lines of evidence: TTR tetramer first dissociates into native monomers, which is shown to be a rate-limiting step in the formation of fibrils. Subsequently, the monomeric species partially unfold to form the aggregation intermediates. Once such intermediates are formed, the following self-assembly process is a downhill polymerization. Hence, tertiary structural changes within the monomers after the dissociation are essential for the amyloid formation. These tertiary structural changes can be facilitated by partial denaturation. To probe the conformational changes under the partially denaturing conditions, five independent trajectories were collected for the wild-type (WT) and its pathogenic variants at 300 and 350 K, resulting in simulations that totaled 59 ns. Under these conditions, L55P variant is more labile than the wild-type and V30M variant. We have observed that the D strand of WT-TTR is trapped in two local minima: the native conformation and the amyloidogenic fold that resembles the surface loop of residues 54-55 of L55P variant. In the tetrameric state, the F strand is bent with large separations at the F-F' interface. This strand becomes flatter in the monomeric state, which may facilitate the formation of new F-F' interface with possible prolonged hydrogen bonds and/or shift in beta-strand register in the fibril state. During the unfolding process, the anticorrelated motion between the strands H and G as well as the strands H and A pulls the H strand out of the inner sheet plane, leading to a more twisted inner sheet. Our simulation has provided important detailed structural information about the partially unfolded state of TTR that may be related to the amyloidogenic intermediates.     

Enhanced erythropoietin heterogeneity in a CHO culture is caused by proteolytic degradation and can be eliminated by a high glutamine level

The molecular heterogeneity of recombinant humanerythropoietin (EPO) increased during the course of abatch culture of transfected Chinese hamster ovary(CHO) cells grown in serum-free medium. This wasshown by both an increased molecular weight and pIrange of the isolated EPO at the end of the culture. However, analysis of the N-glycan structures of themolecule by fluorophore-assisted carbohydrateelectrophoresis (FACE) and HPLC anion exchangechromatography indicated a consistent pattern ofglycosylation. Seven glycoforms were identified, thepredominant structure being a fully sialylatedtetra-antennary glycan. The degree of sialylationwas maintained throughout the culture. Analysis ofthe secreted EPO indicated a time-dependent increasein the molecular weight band width of the peptideconsistent with proteolytic degradation. A highglutamine concentration (16–20 mM) in the culturedecreased the apparent degradation of the EPO.     

The denatured state of Engrailed Homeodomain under denaturing and native conditions

Protein folding starts from the elusive form of the denatured state that is present under conditions that favour the native state. We have studied the denatured state of Engrailed Homeodomain (En-HD) under mildly and strongly denaturing conditions at the level of individual residues by NMR and more globally by conventional spectroscopy and solution X-ray scattering. We have compared these states with a destabilized mutant, L16A, which is predominantly denatured under conditions where the wild-type is native. This engineered denatured state, which could be directly studied under native conditions, was in genuine equilibrium with the native state, which could be observably populated by changing the conditions or introducing a stabilizing mutation. The denatured state had extensive native secondary structure and was significantly compact and globular. But, the side-chains and backbone were highly mobile. Non-cooperative melting of the residual structure on the denatured state of En-HD was observed, both at the residue and the molecular level, with increasingly denaturing conditions. The absence of a co-operative transition could result from the denatured state ensemble progressing through a series of intermediates or from a more general slide (second-order transition) from the compact form under native conditions to the more extended at highly denaturing conditions. In either case, the starting point for folding under native conditions is highly structured and already poised to adopt the native structure.     

Iionization of tyrosyl groups of ovalbumin under native and denaturing conditions.

Phenolic titration of ovalbumin was performed in the pH range 7-12 at 30 degrees C and at three ionic strengths viz. 0.033, 0.133 and 0.200. The conformational integrity of ovalbumin was studied by viscosity measurements at different pH values in the pH range 7-12.4. At ionic strength 0.133 two phenolic groups titrated reversibly with pKint = 10.31, and w = 0.032 up to pH 11.25 under native conditions. The value of w expectedly decreased with increase in ionic strength. Two additional phenolic groups became available for reversible titration between pH 11.25 and 11.95 after some conformational change. Above pH 12, the phenolic titration became irreversible and all of the nine tyrosine residues were titrated at pH 13.3 Exposure of ovalbumin to alkaline pH (12.4) caused considerable disruption of the native protein conformation. The reduced viscosity increased from 4.2 ml/g at pH 7.0 to 16.8 ml/g at pH 12.4 under identical conditions of the protein concentration. All of the nine tyrosyl groups of ovalbumin were titrated normally (pKint = 9.9) in a mixture of 5 M guanidine hydrochloride and 1.2 M urea. However, even in this mixture electrostatic interaction, as measured by w was not completely abolished.     

用变性梯度凝胶电泳分析PCR克隆的突变率

用变性梯度凝胶电泳技术比较分析了分别由Taq DNA聚合酶和Pfu DNA聚合酶催化扩增的产物克隆入pUCm-T/DH5a系统中产生的重组子,发现包含突变的重组子分别为21．50％和3．50％,前为后的6．15倍。转化为每100nt净扩增长度的突变率分别为7．44％和1．21％。     

Detection of laccase activity in polyacrylamide gels after electrophoresis under denaturing conditions

Summary Specific laccase activity was detected on SDS-PAGE using 2,2-Azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid), guaiacol and syringaldazine as substrates. Enzyme activity was detected immediately following electrophoresis, after the detergent diffused from the gel into a renaturation buffer and subsequent Coomassie blue staining. Identification of laccase in a protein mixture and estimation of its molecular weight were done simultaneously.     

用三步法纯化重组人促红细胞生成素

在生物反应器中培养中国仓鼠卵巢细胞—促红细胞生成素(CHO-EPO)C2细胞株,培养上清液中重组人促红细胞生成素(rHuEPO)表达水平达2000～3000U/ml。培养上清经过三步纯化:第一步为反相柱色谱,可将样品浓缩约96.7％,其收集液经充分透析后进行第二步的DEAE-离子交换色谱,最后进行分子筛色谱,总回收率为30％以上,经纯化的rHuEPO比活性为1.5×10~8U/g蛋白,SDSPAGE一条带,扫描测试纯度达98％以上。     

The frequency of bone marrow cells that bind erythropoietin

The frequencies of rat and mouse bone marrow cells capable of binding erythropoietin were studied by both direct fluorescence and indirect immunofluorescence. We found that between 1-2% of the cells bound erythropoietin, that the binding was specific, and that the number of cells that bound erythropoietin was, in part, a function of the erythropoietic state of the donor animal. A statistical method for evaluating the data obtained is included.     

人红细胞生成素受体胞外区克隆及序列测定

人红细胞生成素受体 (h EPOR)是人红细胞生成素的作用配体 ,其胞外区是 h EPO的作用域 ,它的克隆、表达对两种分子的相互作用机制以及 EPO类似物 (新型造血药物 )的筛选都有十分重要的意义。以人胎肝为材料 ,通过对其总 RNA的提取 ,利用 RT- PCR方法扩增 h EPOR的胞外区基因和跨膜区基因及推导相应的氨基酸残基排列 ,结果与国外文献报道相比较从而检验其正确性。     

不同压力条件下油藏内源细菌群落激活过程中变性梯度凝胶电泳分析?

摘要：【目的】了解常压（1 MPa）和高压（10 MPa）条件下内源细菌激活中的细菌群落和结构的变化。【方法】利用胜利油田沾3×24井产出液水样,在常压和高压下进行富集培养后,定时取样,样品用变性梯度凝胶电泳（denature gradient gel electrophoresis,DGGE）技术分析。【结果】通过对内源微生物激活前后DGGE条带的数量和亮度变化分析表明,油藏环境中的细菌在种类和数量上并不丰富,激活剂的加入改变了菌群原有的贫营养环境,从而使一些因营养缺乏生长受抑制细菌得以大量繁殖,菌群结     

Isolation and characterization of a subset of erythropoietin glycoforms with cytoprotective but minimal erythropoietic activity

Although historically used for the treatment of anemia, erythropoietin (EPO) has emerged as a neurotrophic and neuroprotective agent in different conditions of neuronal damage (traumatic brain injury, ischemia, spinal cord compression, peripheral neuropathy, retinal damage, epilepsy, Parkinson's Disease, among others). Nonetheless, EPO's therapeutic application is limited due to its hematological side‐effects. With the aim of obtaining EPO derivatives resembling the hormone isolated from cells and tissues of neural origin, a novel combination of less acidic EPO glycoforms ‐designated as neuroepoetin (rhNEPO)‐ was purified to homogeneity from the supernatant of a CHO‐producing cell line by a four‐step chromatographic procedure. This simple and single process allowed us to prepare two EPO derivatives with distinct therapeutic expectations: the hematopoietic version and a minimally hematopoietic, but mainly in vitro cytoprotective, alternative. Further biological characterization showed that the in vivo erythropoietic activity of rhNEPO was 25‐times lower than that of rhEPO. Interestingly, using different in vitro cytoprotective assays we found that this molecule exerts cytoprotection equivalent to, or better than, that of rhEPO in cells of neural phenotype. Furthermore, despite its shorter plasma half‐life, rhNEPO was rapidly absorbed and promptly detected in the cerebrospinal fluid after intravenous administration in rats (5 min postinjection, in comparison with 30 min for rhEPO). Therefore, our results support the study of neuroepoetin as a potential drug for the treatment of neurological diseases, combining high cytoprotective activity with reduced hematological side‐effects. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Isoelectric focusing and two-dimensional electrophoresis of tubulin using immobilized pH gradients under denaturing conditions

Modifications of the LKB Immobiline isoelectric focusing (IEF) technique are described for use under conditions that solubilize and denature most proteins (8 M urea and 2% Nonidet-P40). This procedure permits pH gradients that are four- to fivefold shallower than previously available with conventional ampholine-IEF procedures. It can also be used as a first dimension in two-dimensional gel electrophoresis. The advantage of the stable ultranarrow pH gradient is demonstrated by directly comparing the resolution of vertebrate brain tubulins using (i) denaturing conventional ampholine-IEF and (ii) denaturing Immobiline-IEF. Analysis of tubulin on the Immobiline-IEF gel increases the separation distance between the individual tubulins and distinguishes differences among tubulin samples that could not be resolved by conventional ampholine isoelectric focusing.