Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice

Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgeulc animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein （EGFP） transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibody preparation.     

Imidazole as a catalyst for in vitro refolding of enhanced green fluorescent protein

Imidazole is a reagent widely used in protein purifying processes. Here, we reveal a novel chaperone-like activity for imidazole using enhanced green fluorescent protein (EGFP) as a model protein. Experimental results showed that imidazole acted as an effective catalyst for refolding of the chemically denatured EGFP and suppressor for the heat-induced aggregation of EGFP. The refolding kinetics was determined in real time. Both the recovering yield and refolding rate of denatured EGFP in the presence of imidazole were increased. The studies on elucidating the mechanism show that imidazole may catalyze the prolyl cis/trans isomerization and the possible mechanism was discussed. To our knowledge, there are no data on the effect of imidazole on protein folding. Considering the prolyl isomerization is the rate-limited step for refolding of most proteins and aggregation is a universal serious problem for biotechnology, imidazole thus represents a previous unknown type of protein-folding catalyst.     

Transgenic mice with pancellular enhanced green fluorescent protein expression in primitive hematopoietic cells and all blood cell progeny

Transgenic mice homogeneously expressing enhanced green fluorescence protein (EGFP) in primitive hematopoietic cells and all blood cell progeny, including erythrocytes and platelets, have not been reported. Given previous data indicating H2Kb promoter activity in murine hematopoietic stem cells (HSCs), bone marrow (BM), and lymphocytes, an H2Kb enhancer/promoter EGFP construct was used to generate transgenic mice. These mice demonstrated pancellular EGFP expression in both primitive BM Sca-1+Lin-Kit+ cells and side population (SP) cells. Additionally, all peripheral blood leukocytes subsets, erythrocytes, and platelets uniformly expressed EGFP strongly. Competitive BM transplantation assays established that transgenic H2Kb-EGFP HSCs had activity equivalent to wildtype HSCs in their ability to reconstitute hematopoiesis in lethally irradiated mice. In addition, immunohistochemistry revealed EGFP transgene expression in all tissues examined. This transgenic strain should be a useful reagent for both murine hematopoiesis studies and functional studies of specific cell types from particular tissues.     

Identification of a functional nuclear export signal in the green fluorescent protein asFP499

The green fluorescent protein (GFP) asFP499 from Anemonia sulcata is a distant homologue of the GFP from Aequorea victoria. We cloned the asFP499 gene into a mammalian expression vector and showed that this protein was expressed in the human lymphoblast cell line Ramos RA1 and in the embryonic kidney 293T cell line (HEK 293T). In HEK 293T cells, asFP499 was localized mainly in the cytoplasm, suggesting that the protein was excluded from the nucleus. We identified (194)LRMEKLNI(201) as a candidate nuclear export signal in asFP499 and mutated the isoleucine at position 201 to an alanine. Unlike the wildtype form, the mutant protein was distributed throughout the cytoplasm and nucleus. This is the first report of a GFP that contains a functional NES.     

衣原体蛋白的亚细胞定位

沙眼衣原体(Chlamydia trachomatis,CT)是一种严格细胞内寄生、有独特发育周期的原核细胞型微生物.CT在宿主细胞浆内增殖,形成光镜可见的典型细胞内包涵体,包涵体为CT在宿主细胞内的生长繁殖提供屏障保护,同时也是CT与宿主细胞进行物质交换和信息传递的门户,CT不仅可从宿主细胞摄取营养物质,还可分泌效应蛋白进入宿主细胞质调节宿主细胞功能.CT基因组DNA序列和功能注释完成后,衣原体蛋白的亚细胞定位、结构和功能的研究已成为衣原体研究领域的热点之一[1-3].在CT与宿主细胞相互作用过程中,Inc蛋白、分泌蛋白等衣原体蛋白可能发挥着重要作用,鉴于蛋白质的亚细胞定位情况往往与其功能密切相关,衣原体蛋白在感染细胞中的定位认识成为其功能研究中的重要环节.     

Development of an assay for beta-lactam hydrolysis using the pH-dependence of enhanced green fluorescent protein

An assay has been developed utilizing the pH-dependent fluorescence of enhanced green fluorescent protein (EGFP). This photoprotein allows for the study of kinetic properties of hydrolytic enzymes based on the production of protons. As a model system, beta-lactamase, a well-characterized enzyme responsible for antibiotic resistance in many bacteria, was used. More specifically, EGFP and beta-lactamase were genetically fused using overlap extension PCR and incorporated into a bacterial expression vector. The vector was subsequently transformed into Escherichia coli, and the fusion protein was expressed and purified. beta-Lactamase catalyzes the hydrolysis of the beta-lactam ring of ampicillin. This causes a decrease in the local pH, which in turn changes the spectral properties of EGFP. This property was utilized to perform enzyme kinetic studies on the new fusion protein as well as on the beta-lactamase inhibitor, sulbactam. The assay can be used to evaluate substrates and inhibitors of beta-lactamase in a format that should be amenable to high-throughput screening.     

植物蛋白质亚细胞定位相关研究概述

蛋白质在植物细胞内的定位是了解蛋白质功能、 基因调控和蛋白质-蛋白质相互作用的关键.近年来随着各种蛋白质亚细胞定位方法的快速发展和技术的不断提升,蛋白质亚细胞定位实现了高通量、活体动态研究.本文总结了植物蛋白质亚细胞定位的常用技术,以及常用细胞器特异性标记的研究进展,并对此领域研究的发展前景做出了展望.     

烟曲霉蛋白质分泌载体的构建及酸性磷酸酯酶的细胞定位

[目的]在酵母细胞中蛋白质的糖基磷酸肌醇化(GPI)修饰是将GPI定位于细胞膜或细胞壁的信号.目前已对酵母GPI蛋白的细胞定位信号有一定了解,但对丝状真菌GPI蛋白的定位则了解甚少.AfPhoA是丝状真菌烟曲霉(Aspergillus fumigatus)的酸性磷酸酯酶,是GPI修饰的蛋白.该蛋白首先分离自细胞膜,随后又发现该蛋白与细胞壁结合.分析其C-端序列也未发现已知的定位信号,因此目前还不能确定其细胞定位.[方法]我们以绿色荧光蛋白(GFP)作为报告分子,将AfPhoA的C-端序列与GFP的C-端融合后检测融合GFP的细胞定位.[结果]我们用烟曲霉几丁质酶AfChiB1的启动子和N-端信号肽构建了可在烟曲霉中分泌表达GFP的表达载体pchiGFP.在此基础上将AfPhoA的C-端与GFP融合,融合质粒与pCDA14共转化烟曲霉后筛选到一株转化子.该转化子可表达融合GFP,在诱导和非诱导条件下,融合GFP均主要分布在细胞膜上,随培养时间的延长,融合GFP在细胞壁上也有少量分布;在培养上清液中只能检出约30KD的GFP融合蛋白,而没有完整的GFP融合蛋白,推测为从GPI锚上水解释放的.[结论]我们的研究结果表明,AfPhoA蛋白GPI修饰的作用是使该蛋白定位于细胞膜.本研究不仅初步确定了AfPhoA蛋白GPI修饰的细胞膜定位功能,而且为烟曲霉基因与蛋白质功能的研究建立了一个有效表达系统.     

辅助病毒依赖型腺病毒载体转基因的体外表达效率

构建可表达增强型绿色荧光蛋白 (Enhanced green fluorescent protein,EGFP) 的辅助病毒依赖型腺病毒载体 (Helper-dependent adenoviral vector,HDAd),并完成大量制备、纯化和体外表达鉴定。荧光显微镜证实HDAd/EGFP可表达,电镜下观察到经CsCl纯化后的腺病毒的典型形态。分光光度计法测定病毒的浓度为4.0×1012 颗粒数 (Virus particle,vp) /mL。与可表达EGFP的第一代腺病毒载体 (First generation adenoviral vector,FGAd) FGAd/EGFP进行了体外感染和转基因表达效率的比较研究,分别用约2 000 vp/细胞的HDAd/EGFP和FGAd/EGFP感染A549细胞,流式细胞仪检测EGFP的表达情况。通过相同时间点流式细胞仪分析EGFP的表达情况,可见HDAd/EGFP感染早期的A549细胞较FGAd/EGFP有更高的荧光表达率及更高的表达强度,显示HDAd载体具有转基因瞬时高表达的特性,是一种更有价值的疫苗载体。     

Subcellular localization of the Schlafen protein family

Although the first members of the Schlafen gene family were first described almost 10 years ago, the precise molecular/biochemical functions of the proteins they encode still remain largely unknown. Roles in cell growth, haematopoietic cell differentiation, and T cell development/maturation have, with some experimental support, been postulated, but none have been conclusively verified. Here, we have determined the subcellular localization of Schlafens 1, 2, 4, 5, 8, and 9, representing all three of the murine subgroups. We show that the proteins from subgroups I and II localize to the cytoplasm, while the longer forms in subgroup III localize exclusively to the nuclear compartment. We also demonstrate upregulation of Schlafen2 upon differentiation of haematopoietic cells and show this endogenous protein localizes to the cytoplasm. Thus, we propose the different subgroups of Schlafen proteins are likely to have functionally distinct roles, reflecting their differing localizations within the cell.     

沙眼衣原体CT058蛋白在感染细胞中的定位分析

分析沙眼衣原体CT058蛋白在感染细胞中的定位.克隆表达CT058蛋白;纯化的CT058融合蛋白免疫小鼠制备多克隆抗体;间接免疫荧光法对CT058蛋白在沙眼衣原体感染细胞中的定位进行分析;Western blot检测CT058蛋白在原体和网状体中的表达情况.间接免疫荧光染色实验显示CT058蛋白位于包涵体内;鼠抗GST-CT058抗体与GST-CT058融合蛋白吸附后特异性染色消失,而与GST-CT232融合蛋白吸附后仍然可见GST-CT058抗体的包涵体染色特征;Western blot证实CT058蛋白在纯化的原体和网状体上均有表达.CT058蛋白定位于沙眼衣原体感染细胞的包涵体内.     

Transgene expression of enhanced green fluorescent protein in cloned rabbits generated from in vitro-transfected adult fibroblasts

Live rabbits have previously been generated through nuclear transfer using adult cells as nuclear donors. We demonstrated in this study that transfected adult rabbit fibroblasts are also capable of supporting full-term development. The fibroblasts were transfected with a pEGFP-C1 plasmid using lipofectamine^™ 2000, and the transgenic cells were derived from conditioned medium. The transgenic fibroblasts were cultured until confluent and then serum-starved prior to be used as nuclear donors. After nuclear transfer and activation, 22% (12/55) of the transgenic cloned embryos developed to the blastocyst stage. A total of 114 embryos at the 4- to 8-cell stage were transferred to the oviducts of 8 pseudo-pregnant mothers; 5 of these animals became pregnant, and 3 of the 5 mother rabbits carried the pregnancy to term. Caesarean section was performed on the 3 pregnant mothers, yielding 4 kits, one of which has survived for more than 9 months. Green fluorescence could be detected in the toenails of the living cloned rabbit and the offspring from the living cloned rabbit under ultraviolet light. DNA analyses confirmed that all 4 cloned rabbits were genetically identical to the transgenic donor cells, and that they all carried the EGFP gene. The present study demonstrated that transgenic rabbits can be generated through nuclear transfer. These results may facilitate future developments in the genetic engineering of rabbits.     

Expression of enhanced green fluorescent protein in porcine- and bovine-cloned embryos following interspecies somatic cell nuclear transfer of fibroblasts transfected by retrovirus vector

Interspecies somatic cell nuclear transfer (iSCNT) has emerged as an important tool for studying nucleo-cytoplasmic interactions and cloning of animals whose oocytes are difficult to obtain. This study was designed to explore the feasibility of employing transgenic fibroblasts as donor cells for iSCNT. The study examined the chromatin morphology, in vitro development, and expression of an enhanced green fluorescent protein (EGFP) gene in porcine- and bovine-cloned embryos produced by iSCNT of fetal fibroblast transfected with a pLNbeta-EGFP retroviral vector. Parthenogenetic and transfected or nontransfected intraspecies SCNT embryos were used as controls for comparison. Analysis of data revealed that xenogenic oocyte was able to reprogram somatic cells of different genus and supports their in vitro development to the blastocyst stage. However, the developmental rates of transgenic iSCNT embryos to the blastocyst stage were significantly lower than those of intraspecies SCNT embryos. The reduction in development rates was however, not due to integration of the transgene as the lower (P < 0.05) development rates of the intraspecies SCNT porcine or bovine embryos did not differ between transgenic and nontransgenic groups. Expression of EGFP was observed in 100% of blastocysts and mosaicism was not observed. Furthermore, after iSCNT of porcine or bovine donor nuclei into xenogenic ooplasm, patterns of nuclear remodeling in reconstructed embryos were similar. In conclusion, our data demonstrated the feasibility of producing transgenic iSCNT embryos. To our knowledge, this is the first report of transgenic cloned embryo production by iSCNT approach. In the future, this may provide a powerful research tool for studying developmental events in domestic animals and provide marked cell lines for other genetic manipulations.     

Identification of the subcellular localization of mycobacterial proteins using localization motifs

Mycobacterium, the most common disease-causing genus, infects billions of people and is notoriously difficult to treat. Understanding the subcellular localization of mycobacterial proteins can provide essential clues for protein function and drug discovery. In this article, we present a novel approach that focuses on local sequence information to identify localization motifs that are generated by a merging algorithm and are selected based on a binomially distributed model. These localization motifs are employed as features for identifying the subcellular localization of mycobacterial proteins. Our approach provides more accurate results than previous methods and was tested on an independent dataset recently obtained from an experimental study to provide a first and reasonably accurate prediction of subcellular localization. Our approach can also be used for large-scale prediction of new protein entries in the UniportKB database and of protein sequences obtained experimentally. In addition, our approach identified many local motifs involved with the subcellular localization that also interact with the environment. Thus, our method may have widespread applications both in the study of the functions of mycobacterial proteins and in the search for a potential vaccine target for designing drugs.     

Identification of an unconventional nuclear localization signal in human ribosomal protein S2

Ribosomal proteins must be imported into the nucleus after being synthesized in the cytoplasm. Since the rpS2 amino acid sequence does not contain a typical nuclear localization signal, we used deletion mutant analysis and rpS2-beta-galactosidase chimeric proteins to identify the nuclear targeting domains in rpS2. Nuclear rpS2 is strictly localized in the nucleoplasm and is not targeted to the nucleoli. Subcellular localization analysis of deletion mutants of rpS2-beta-galactosidase chimeras identified a central domain comprising 72 amino acids which is necessary and sufficient to target the chimeric beta-galactosidase to the nucleus. The nuclear targeting domain shares no significant similarity to already characterized nuclear localization signals in ribosomal proteins or other nuclear proteins. Although a Nup153 fragment containing the importinbeta binding site fused to VP22 blocks nuclear import of rpS2-beta-galactosidase fusion proteins, nuclear uptake of rpS2 could be mediated by several import receptors since it binds to importinalpha/beta and transportin.