Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system

A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 10⁴ molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.     

FRAP法对内源性GFP在活细胞中动态分布的共焦显微镜成像

各种分子在核质问的动态分布与它们的跨膜转运密切相关。离子、r证矾A和多数小分子量蛋白可以通过核孔复合物（NPG,nuclear pore complexes）在核质问自由扩散,而分子量大于70kDa的分子需要ATP和核定位序列才能实现跨膜转运。本实验利用荧光漂白后恢复（FRAP,fluorescence recovery after photobleaching）法观测人肺腺癌肿瘤细胞（ASTC-a-1）中表达的27 kDa EGFP在核质问的被动扩散,并以激光共焦显微镜进行实时成像。转染EGFP外源基因的肿瘤细胞系在经过半年的传代培养后仍能稳定而高效的表达其荧光标记。实验表明,EGFP分子可以通过核孔在核质间被动扩散,但扩散速度远低于在核内或质内的速度,没有证据表明EGFP可以在细胞问扩散。     

Polydendrocytes display large lineage plasticity following focal cerebral ischemia

Polydendrocytes (also known as NG2 glial cells) constitute a fourth major glial cell type in the adult mammalian central nervous system (CNS) that is distinct from other cell types. Although much evidence suggests that these cells are multipotent in vitro, their differentiation potential in vivo under physiological or pathophysiological conditions is still controversial.To follow the fate of polydendrocytes after CNS pathology, permanent middle cerebral artery occlusion (MCAo), a commonly used model of focal cerebral ischemia, was carried out on adult NG2creBAC:ZEG double transgenic mice, in which enhanced green fluorescent protein (EGFP) is expressed in polydendrocytes and their progeny. The phenotype of the EGFP(+) cells was analyzed using immunohistochemistry and the patch-clamp technique 3, 7 and 14 days after MCAo. In sham-operated mice (control), EGFP(+) cells in the cortex expressed protein markers and displayed electrophysiological properties of polydendrocytes and oligodendrocytes. We did not detect any co-labeling of EGFP with neuronal, microglial or astroglial markers in this region, thus proving polydendrocyte unipotent differentiation potential under physiological conditions. Three days after MCAo the number of EGFP(+) cells in the gliotic tissue dramatically increased when compared to control animals, and these cells displayed properties of proliferating cells. However, in later phases after MCAo a large subpopulation of EGFP(+) cells expressed protein markers and electrophysiological properties of astrocytes that contribute to the formation of glial scar. Importantly, some EGFP(+) cells displayed membrane properties typical for neural precursor cells, and moreover these cells expressed doublecortin (DCX)--a marker of newly-derived neuronal cells. Taken together, our data indicate that polydendrocytes in the dorsal cortex display multipotent differentiation potential after focal ischemia.     

Characterization of an extended receptive ligand repertoire of the human olfactory receptor OR17-40 comprising structurally related compounds

Molecular properties of odorant compounds essential for activation of the human olfactory receptor hOR17-40 were investigated using a collection of 23 variants of its cognate ligand helional. Coupling receptor activation to an optically detectable intracellular Ca(2+) ion flux allowed dose-dependent screening of different odorant molecules in human embryonic kidney (HEK)293 cells. We found an extended collection of activating ligands and provide first evidence for hOR17-40-specific antagonists. The C-terminal fusion of enhanced green fluorescent protein to the hOR17-40 retained full receptor function and permitted the selection of cells with defined receptor expression levels, which was an essential step for optimizing our screening protocol. Interestingly, cells with a low EGFP fluorescence intensity exhibited efficient hOR17-40 cell surface targeting and odorant-evoked signal transduction; in contrast, highly fluorescent cells displayed mainly incorrectly targeted, intracellular receptors. Fluorescence-activated cell sorting was used to separate hOR17-40-expressing cells on the basis of their endogenous EGFP fluorescence intensity, thereby increasing the fraction of odorant-responsive cells to up to 80% of the total cell number.     

Organophosphorus compound detection on a cell chip with yeast coexpressing hydrolase and eGFP

Organophosphorus compounds (OPs) such as pesticides, fungicides, and herbicides are highly toxic but are nevertheless extensively used worldwide. To detect OPs, we constructed a yeast strain that co-displays organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (EGFP) on the cell surface using a Flo1p anchor system. OP degradation releases protons and causes a change in pH. This pH change results in structural deformation of EGFP, which triggers quenching of its fluorescence, thereby making this cell useful for visual detection of OPs. Fluorescence microscopy confirmed the high-intensity fluorescence displayed by EGFP on the cell surface. The yeast strain possessed sufficient OPH hydrolytic activities for degrading OPs, as measured by incubation with 1 mM paraoxon for 24 h at 30°C. In addition, with 20 mM paraoxon at 30°C, fluorescence quenching of EGFP on the single yeast cell was observed within 40 s in a microchamber chip. These observations suggest that engineered yeast cells are suitable for simultaneous degradation and visual detection of OPs.     

Expression of SLAM (CDw150) on Sf9 cell surface using recombinant baculovirus mediates measles virus infection in the nonpermissive cells

Signaling lymphocytic activation molecule (SLAM; also known as CDw150) has been reported as the receptor of measles virus (MV) interacting with MV hemagglutinin (MVH). In this study, we developed a baculovirus-derived vector, the Bacmid-egfp, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP) under the control of the promoter of very late polyhedrin gene from Autographa californica multiple nucleopolyhedrovirus (AcMNPV), and employed the recombinant baculovirus to express SLAM in Sf9 (Spodoptera frugiperda) cells and investigate SLAM function. The result showed that the integration of the EGFP expression cassette in the Bac-to-Bac system facilitated research with the system without introducing compromises due to its use. SLAM protein fused to His-tag was expressed in Sf9 cells through the modified Bac-to-Bac system. The expressed SLAM was identified as approximately 46 kDa, and it presented on the cell surface, as revealed by fluorescent immunochemical staining and confocal microscopic analysis. The pull-down assay proved that SLAM protein expressed in this system could interact with MVH protein. After incubating with MV vaccine strain S191, cell fusion was only observed in the Sf9 cells expressing both EGFP and SLAM from recombinant baculovirus rather than those expressing EGFP only from the modified viral vector. Furthermore, MV replicated and induced apoptosis in the Sf9 cells with SLAM expression.     

Specific protein delivery to target cells by antibody-displaying bionanocapsules

Bionanocapsules (BNCs) are nanoparticles with a high biocompatibility composed of the L protein of the hepatitis B virus surface antigen. BNC can deliver bioactive molecules to hepatocytes efficiently and specifically. However, delivery is limited to hepatocytes and incorporation of proteins into BNC is quite troublesome. Here, in order to alter the specificity of BNC and to achieve efficient protein delivery, we developed engineered BNC displaying the ZZ domain of protein A and incorporating enhanced green fluorescent protein (EGFP) inside the particles using an insect cell expression system. The ZZ domain displayed on the surface of BNC binds to anti-epidermal growth factor receptor (EGFR) antibodies, allowing specific delivery of EGFP to HeLa cells. The engineered BNCs are a promising and powerful tool for efficient and cell-specific protein delivery.     

乙型肝炎病毒蛋白和绿色荧光蛋白的共表达研究

目的：构建增强型绿色荧光蛋白（EGFP）标记的乙型肝炎病毒（HBV）真核表达载体,并研究其在真核细胞和小鼠体内的共表达。方法：以质粒pBR322-HBVadr2．0和pCX-EGFP为基础,构建含有双拷贝HBV全基因组DNA和EGFP基因的真核表达载体pCX-EGFP-HBVadr2．0,分别转染真核细胞和小鼠肝组织,建立体外、体内表达系统,研究GFP和HBV基因的表达。结果：构建了真核表达载体pCX-EGFP-HBVadr2．0,EGFP和HBV病毒蛋白在体内和体外均可表达。结论：构建的pCX-EGFP-HBVadr2．0真核表达载体可以GFP作为HBV存在与否的报告基因,提高了培育检测转基因小鼠的效率,为转基因小鼠的制备及后续研究奠定了基础。     

Temporally controlled site-specific mutagenesis in the germ cell lineage of the mouse testis

We have obtained a PrP-Cre-ER(T) transgenic mouse line (28.8) that selectively expresses in testis the tamoxifen-inducible Cre-ER(T) recombinase under the control of a mouse Prion protein (PrP) promoter-containing genomic fragment. Cre-ER(T) is expressed in spermatogonia and spermatocytes, but not in Sertoli and Leydig cells. We also established reporter PrP-L-EGFP-L transgenic mice harboring a LoxP-flanked enhanced green fluorescent protein (EGFP) Cre reporter cassette under the control of the same PrP promoter-containing genomic fragment that exhibits prominent EGFP expression in brain and testis. Using the PrP-L-EGFP-L as well as other Cre-reporter mice, we demonstrate that tamoxifen administration efficiently and selectively induces Cre-mediated recombination in the germ cell lineage. The established PrP-Cre-ER(T) line should provide a valuable tool for studying functions of germ cell-expressed genes involved in spermatogenesis.     

Functional cell surface display and controlled secretion of diverse Agarolytic enzymes by Escherichia coli with a novel ligation-independent cloning vector based on the autotransporter YfaL

Autotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) of Escherichia coli for the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes from Saccharophagus degradans 2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 10(4) molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growing E. coli.     

Simultaneous analysis of the cyan, yellow and green fluorescent proteins by flow cytometry using single-laser excitation at 458 nm.

BACKGROUND: Development of spectrally distinct green fluorescent protein (GFP) variants has allowed for simultaneous flow cytometric detection of two different colored mutants expressed in a single cell. However, the dual-laser methods employed in such experiments are not widely applicable since they require a specific, expensive laser, and single-laser analysis at 488 nm exhibits considerable spectral overlap. The purpose of this work was to evaluate detection of enhanced cyan fluorescent protein (ECFP) in combination with the enhanced green (EGFP) and enhanced yellow (EYFP) fluorescent proteins by flow cytometry. METHODS: Cells transfected with expression constructs for EGFP, EYFP, or ECFP were analyzed by flow cytometry using excitation wavelengths at 458, 488, or 514 nm. Fluorescence signals were separated with a custom optical filter configuration: 525 nm shortpass and 500 nm longpass dichroics; 480/30 (ECFP), 510/20 (EGFP) and 550/30 (EYFP) bandpasses; 458 nm laser blocking filters. RESULTS: All three fluorescent proteins when expressed individually or in combination in living cells were excited by the 458 nm laser line and their corresponding signals could be electronically compensated in real time. CONCLUSIONS: This method demonstrates the detection of three fluorescent proteins expressed simultaneously in living cells using single laser excitation and is applicable for use on flow cytometers equipped with a tunable argon ion laser.     

A high-throughput screening of genes that encode proteins transported into the endoplasmic reticulum in mammalian cells

The compartments of eukaryotic cells maintain a distinct protein composition to perform a variety of specialized functions. We developed a new method for identifying the proteins that are transported to the endoplasmic reticulum (ER) in living mammalian cells. The principle is based on the reconstitution of two split fragments of enhanced green fluorescent protein (EGFP) by protein splicing with DnaE from Synechocystis PCC6803. Complementary DNA (cDNA) libraries fused to the N-terminal halves of DnaE and EGFP are introduced in mammalian cells with retroviruses. If an expressed protein is transported into the ER, the N-terminal half of EGFP meets its C-terminal half in the ER, and full-length EGFP is reconstituted by protein splicing. The fluorescent cells are isolated using fluorescence-activated cell sorting and the cDNAs are sequenced. The developed method was able to accurately identify cDNAs that encode proteins transported to the ER. We identified 27 novel proteins as the ER-targeting proteins. The present method overcomes the limitation of the previous GFP- or epitope-tagged methods, using which it was difficult to identify the ER-targeting proteins in a high-throughput manner.     

绵羊胎儿成纤维细胞体外培养及转基因研究

目的用增强型绿色荧光蛋白(EGFP)基因转染体外培养绵羊胎儿成纤维细胞,探讨绿色荧光蛋白对绵羊胎儿成纤维细胞生物学特性的影响.方法体外分离培养绵羊胎儿成纤维细胞,经脂质体介导EGFP基因转染第一代成纤维细胞,G418筛选10～12*!d,挑选转基因单克隆细胞,传代培养,进行细胞形态观察、生长曲线以及染色体核型分析,并进行了培养细胞性别鉴定.结果整合有EGFP基因的绵羊胎儿成纤维细胞生物学行为与未转染外源基因的细胞无明显差别,根据荧光强度可直接反应外源基因的表达量.结论 EGFP基因作为体内报告基因可用于转基因细胞的研究,并将整合有EGFP基因的转基因细胞为克隆动物提供核供体奠定了基础.     

A null mutation for tissue inhibitor of metalloproteinases-3 (Timp-3) impairs murine bronchiole branching morphogenesis

Juvenile zebrafish are hermaphroditic; undifferentiated gonads first develop into ovary-like tissues, which then either become ovaries and produce oocytes (female) or degenerate and develop into testes (male). In order to fully capture the dynamic processes of germ cells' proliferation and juvenile hermaphroditism in zebrafish, we established transgenic lines TG(beta-actin:EGFP), harboring an enhanced green fluorescent protein (EGFP) gene driven by a medaka beta-actin promoter. In TG(beta-actin:EGFP), proliferating germ cells and female gonads strongly expressed EGFP, but fluorescence was only dimly detected in male gonads. Based on the fluorescent (+) or nonfluorescent (-) appearance of germ cells seen in living animals, three distinct groups were evident among TG(beta-actin:EGFP). Transgenics in ++ group (44%) were females, had fluorescent germ cells as juveniles, and female gonads continuously fluoresced throughout sexual maturation. Transgenics in +- (23%) and -- (33%) groups were males. Fluorescent germ cells were transiently detected in +- transgenics from 14 to 34 days postfertilization (dpf), but were not detected in -- transgenics throughout their life span. Histological analyses showed that 26-dpf-old transgenics in ++, +-, and -- groups all developed ovary-like tissues: Germ cells in -- group juveniles arrested at the gonocyte stage and accumulated low quantities of EGFP, while those in ++ group juveniles highly proliferated into diplotene to perinucleolar stages and accumulated high quantities of EGFP. In +- group juveniles, degenerating oocytes, gonocytes, and spermatogonia were coexistent in transiently fluorescent gonads. Therefore, the fluorescent appearance of gonads in this study was synchronous with the differentiation of ovary-like tissues. Thus, TG(beta-actin:EGFP) can be used to visualize germ cells' proliferation and juvenile hermaphroditism in living zebrafish for the first time.     

GFP-SA融合蛋白的表达纯化及其锚定修饰肿瘤细胞的研究

目的　GFP（绿色荧光蛋白）-SA（链亲和素）双功能融合蛋白的制备及其鉴定研究,以展示我们建立的技术平台,即用含链亲和素的双功能融合蛋白对生物素化的细胞表面进行高效的锚定修饰。方法　构建原核表达载体pET24d/GFP-SA转化大肠杆菌BL21(DE3)。用IPTG诱导重组蛋白的表达,用镍金属螯合（Ni-NTA）层析柱进行纯化。用制备的GFP-SA双功能融合蛋白,对B16肿瘤细胞已生物素化的细胞表面进行修饰,经荧光显微镜和流式细胞仪进行修饰效率分析。此外,用MTT法检测细胞表面修饰对肿瘤细胞活力及其生长情况的影响。结果　GFP-SA重组融合蛋白在大肠杆菌实现了高效表达（约占细菌总蛋白的20%）,通过纯化和复性制备的GFP-SA双功能融合蛋白具有双重活性,即：链亲和素介导的、对生物素高效特异的结合活性,和GFP发射绿色荧光的活性,并能高效修饰表面已生物素化的肿瘤细胞。此外,GFP-SA双功能融合蛋白的细胞表面修饰对细胞的活力及其生长无显著影响。结论　GFP-SA融合蛋白能高效修饰表面已生物素化的肿瘤细胞,可用作肿瘤疫苗研究的示踪蛋白及实验对照体系。     

An expression system for screening of proteins for glycan and protein interactions

Here we describe a versatile high-throughput expression system that permits genome-wide screening of type 1 membrane and secreted proteins for interactions with glycans and proteins using both cell-expressed and soluble forms of the expressed proteins. Based on Gateway cloning methodology, we have engineered a destination vector that directs expression of enhanced green fluorescent protein (EGFP)-tagged proteins at the cell surface via a glycosylphosphatidylinositol tail. The EGFP fusion proteins can then be cleaved with PreScission protease to release soluble forms of proteins that can be optionally biotinylated. We demonstrate the utility of this cloning and expression system for selected low-affinity membrane lectins from the siglec family of sialic acid-binding immunoglobulin-like lectins, for the glycosaminoglycan-binding proteins FGF-1 and BACE, and for the heterotypic adhesion molecules JAM-B and JAM-C. Cell-expressed proteins can be evaluated for glycan interactions using polyvalent soluble glycan probes and for protein interactions using either cells or soluble proteins. Following cleavage from the cell surface, proteins were complexed in solution and sufficient avidity was achieved to measure weak protein–glycan and weak protein–protein interactions using glycan arrays and surface plasmon resonance, respectively.     

The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells

EGFP (enhanced green fluorescent protein) tagged to either the N (amino)-terminus [EGFP/hERG (human ether-a-go-go-related gene)] or C (carboxyl)-terminus (hERG/EGFP) of hERG channel is used to study mutant channel protein trafficking for several years. However, it has been reported that the process can alter hERG channel properties. The aim of the study was to determine whether EGFP tagged to N-terminus of hERG channels would alter the cellular localizations and the electrophysiological properties of hERG channels compared with untagged hERG channels. The hERG channels tagged with or without EGFP were transiently expressed in HEK (human embryonic kidney) 293 cells using a lipofectamine method. HEK 293 cells expressing pCDNA3-hERG or pEGFP-hERG were double immunolabelled with anti-hERG and anti-calnexin (an ER marker protein) followed with FITC- and TRITC (tetramethylrhodamine β-isothiocyanate)-labelled secondary antibodies, respectively. Confocal laser scanning microscope was used to observe the cellular localization of EGFP-tagged hERG channels and untagged hERG channels. Patch-clamp technique was used to record whole cell currents. We found that the EGFP/hERG fusion protein and untagged hERG channels were both expressed not only on the cell surface membrane but also in the cytoplasm of HEK293 cells. The EGFP/hERG appeared to influence the hERG channel gating properties, including reduction of the peak tail current density, more rapid inactivation process, faster recovery from inactivation and faster deactivation kinetics compared with untagged hERG channels. Our results suggest that the EGFP/hERG channel alter the electrophysiological properties of hERG channel, but it does not seem to alter the cellular location of hERG channels. Thus, EGFP tagging to N-terminus might be used for research of subcellular location of hERG channels but not for the channel electrophysiological properties.