HeLa细胞表达分泌重组eGFP-DPF-1在卵母细胞上的定位

将兔输卵管蛋白(DPF-1)基因连结于增强型绿色荧光蛋白(eGFP)基因5′端,构建了真核表达重组质粒(pEGFP-N1/DPF-1),转染HeLa细胞,获得稳定表达分泌融合蛋白eGFP-DPF-1的HeLa细胞株。该融合蛋白呈现的分子量达120 KD,提示经翻译后修饰。取兔卵母细胞-卵丘细胞复合物(COC)、去除卵丘细胞后的卵母细胞或输卵管内的卵母细胞,与该株细胞共培养或培养于该株细胞条件培液中,观察兔输卵管蛋白在兔卵母细胞上的分布。结果显示DPF-1大量结合于卵母细胞透明带,先结合于透明带内层,然后维持在内层多外层少的分布状态上;在卵母细胞质膜表面则呈点状均匀分布。DPF-1在卵母细胞上的分布不受其周围颗粒细胞的阻碍,且颗粒细胞上未见有DPF-1结合的痕迹。本实验首次证实体外真核细胞表达分泌的输卵管蛋白能与卵母细胞结合,并借助绿色荧光蛋白作为示踪信号体外直接观察到该表达产物在卵母细胞上的动态分布,为进一步深入分析输卵管蛋白的功能提供了线索,也为研究输卵管内其他蛋白在配子/早胚上定位提供了可行的办法。     

新的重组蛋白表达系统——植物根分泌和叶分泌

多种基因工程抗体、酶、激素、血浆蛋白和疫苗等都已在植物的叶、茎、根、果实、种子以及植物细胞和器官中得到表达 ,然而提取和纯化始终是大规模利用植物生产重组蛋白的主要障碍 .Ｂｏｒｉｓｊｕｋ和Ｋｏｍａｍｙｔｓｋｙ等 (1999年和 2 0 0 0年 )依据内质网和内质网信号肽在蛋白质合成中的作用 ,把 3种重组蛋白 ,嗜热细菌来源的木聚糖酶、水母的绿色荧光蛋白和人胎盘分泌的碱性磷酸酶 (ＳＥＡＰ) ,定位到质外体中 ,通过植物根分泌和叶分泌途径获得表达 ,从而建立了 2种新的重组蛋白表达系统———植物根分泌和叶分泌 ,简化了分离和纯化程…     

重组蛋白在大肠杆菌周间腔的分泌表达

大肠杆菌是用于生产重组蛋白的重要工程宿主菌。但是,要获得足够的正确折叠的蛋白还存在一定的缺陷,其中一种解决此问题的方法就是使重组蛋白分泌到大肠杆菌的周间腔里。在这篇综述中,主要讨论了使重组蛋白分泌表达至大肠杆菌周间腔的近期的研究进展。     

人PAK1重组蛋白质的表达及在人胃癌BGC-823细胞内定位

为检测人PAK1在原核细胞中的表达情况及在真核细胞中的定位,利用RT-PCR技术获得PAK1目的基因,构建GST融合蛋白原核表达载体pGEX-5X-1/PAK1,IPTG诱导后进行SDS-PAGE及Western印迹检测,并用体外激酶实验测定其生物学活性;同时,构建带绿色荧光蛋白(GFP)标签的真核表达载体pEGFP/PAK1,利用脂质体法转入人胃癌BGC-823细胞系中,在共焦激光扫描显微镜下观察癌细胞中绿色荧光蛋白的表达。结果表明,pGEX-5X-1/PAK1载体能在大肠杆菌BL-21中正确表达GST-PAK1融合蛋白,大小约为94kDa,具有生物活性;PAK1绿色荧光蛋白定位于细胞浆。上述研究为进一步探索PAK1的生物学特性及信号转导通路打下了基础。     

真核表达载体pcDNA3.1(-)+KG的构建及在HeLa细胞中的表达

为了将绿色荧光蛋白(green fluorescent protein,GFP)引入细胞核内,采用两轮PCR方法从原先克隆在pcD-NA3.1(-)+GFP载体中将GFP编码序列扩增出来并引入Kozak序列和核定位信号,使用常规酶切和连接方法将其重组至pUCm-T克隆载体中,再将目的片段重组至pcDNA3.1(-)中,对阳性克隆进行酶切、PCR和测序鉴定后,构建了带有Kozak序列和核定位信号的绿色荧光蛋白(GFP)真核表达载体pcDNA3.1(-)+KG。真核表达载体pcDNA3.1(-)+KG被转染试剂Su-perfect转染至HeLa细胞中,绿色荧光蛋白基因在HeLa细胞中得到表达而且在细胞核中观察到绿色荧光。该研究以绿色荧光蛋白为标记初步建立了活体观察真核细胞核动态变化的研究体系。     

拟南芥血红蛋白1的亚细胞定位

为研究拟南芥的血红蛋白1(AtGLB1)基因的亚细胞定位,该实验构建了拟南芥血红蛋白1基因与绿色荧光蛋白基因融合的植物表达载体pUCGFP/ AtGLB1.利用基因枪转化法将重组载体转入洋葱表皮细胞瞬时表达,通过检测融合蛋白在洋葱表皮细胞中的分布来确定拟南芥血红蛋白1在细胞中的定位.荧光显微镜检测结果表明,AtGLB1基因表达产物主要定位在细胞核中,少量定位在细胞质中.     

PLK3在前列腺癌细胞中的表达与定位

据报道,Polo样蛋白激酶3(polo-like kinase 3,PLK3)具有促进前列腺癌细胞的生长增殖及迁移能力。本研究旨在构建pCDNA3-FLAG-PLK3真核表达质粒,初步探究其在前列腺癌细胞系内的表达与定位,并检测前列腺癌细胞系中的内源PLK3蛋白水平。根据PLK3蛋白的编码序列设计合成PLK3蛋白编码序列的引物,以含有PLK3蛋白编码序列的质粒作为模板,并通过PCR扩增目的片段,再用限制性内切酶EcoRⅠ和XhoⅠ进行双酶切、连接并转化后,挑取单克隆菌落扩增并提取质粒,进行双酶切鉴定以及测序比对。将序列比对正确的重组质粒转染至CWR22Rv1及LNCaP细胞中,利用Western Blot实验检测重组质粒在CWR22Rv1和LNCaP细胞中的表达;以免疫荧光染色实验检测外源的FLAG-PLK3蛋白在细胞中的定位。此外,利用Western Blot实验检测了在五种前列腺癌细胞系中内源PLK3蛋白水平。本研究经过以上实验构建出了FLAG-PLK3真核表达质粒,并且验证其能够在前列腺癌细胞中表达;通过免疫荧光确定FLAG-PLK3蛋白在细胞中主要分布在细胞膜中,少量分布在细胞...     

重组BPI23—Fcγ1融合蛋白在CHO细胞中的表达

The fusion gene of BPI23 and human Fc gamma 1 was obtained by PCR method, and the expression plasmid was constructed to express recombinant BPI23-Fc gamma 1 fusion protein in CHO cells. After transfection with the plasmid and selection by methotrexate, the cell lines expressing the fusion protein were obtained. The recombinant protein was purified using cation-exchange chromatography and its bioactivity was proved with bactericidal assays.     

SLP-2荧光表达载体的构建及其在乳腺癌细胞MCF-7中的表达与定位

人的SLP-2基因是一个新的肿瘤相关基因,它在多个癌组织里高表达,如食管鳞状细胞癌组织和肺癌组织。其中在乳腺癌组织的高表达和病人的存活率负相关,这说明该基因很可能在乳腺癌的发生中发挥重要作用。为了研究SLP-2基因在肿瘤里的功能,将人类SLP-2基因全长编码区定向连入pEGFP—C3质粒,使SLP-2蛋白可以与绿色荧光蛋白在乳腺癌细胞MCF-7内融合表达。而且细胞荧光实验发现SLP-2蛋白主要定位在MCF-7细胞的胞质内,这为进一步研究SLP-2基因在乳腺癌中的功能奠定了实验基础。     

Purification and characterization of the glycoprotein hormone alpha-subunit-like material secreted by HeLa cells

The protein secreted by HeLa cells that cross-reacts with antiserum developed against the alpha-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 10(5) ng of alpha/mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-alpha had a composition very similar to that of the urinary hCG alpha-subunit. Peptide fingerprints of the HeLa protein and hCG-alpha revealed that several of the Tyr-, Met-, and Cys-containing tryptic peptides were held in common, thus identifying the tumor protein as a glycoprotein hormone alpha-subunit with a primary structure similar to that of hCG-alpha. However, comparison of hCG-alpha and HeLa-alpha demonstrated that the tumor-associated subunit was not identical with its normal counterpart. Only two of the three Tyr-containing tryptic peptides present in hCG-alpha could be detected in HeLa-alpha after iodination with 125I. HeLa-alpha eluted prior to hCG-alpha during Sephadex G-75 chromatography, but the subunits coeluted when the tumor protein was first subjected to mild acid hydrolysis. The purified tumor protein had an apparent molecular weight greater than that of the urinary alpha-subunit when analyzed by SDS-PAGE (Coomassie blue staining), and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI (4.7-5.5 compared to 6.5-7.8), and removal of sialic acid by mild acid hydrolysis did not entirely eliminate this difference. Immunoprecipitation and electrophoresis of alpha-subunit from HeLa cultures labeled with [3H]fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-alpha hydrolysates by HPLC confirmed previous reports that the placental subunit does not contain fucose. HeLa alpha-subunit was unable to combine with hCG beta-subunit to form holo-hCG under conditions where the hCG alpha-subunit was able to do so. The results indicate that, regardless of whether or not a single alpha-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors     

Characterization of active barley alpha-amylase 1 expressed and secreted by Saccharomyces cerevisiae

Recombinant barley -amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified -amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The k _cat/K _m was 2.7 × 10² mM^–1.s^–1, consistent with those of -amylases from plants and other sources.     

FDC-SP,a novel secreted protein expressed by follicular dendritic cells

To define better the molecular basis for follicular dendritic cell (FDC) function, we used PCR-based cDNA subtraction to identify genes specifically expressed in primary FDC isolated from human tonsils. In this work we report the discovery of a novel gene encoding a small secreted protein, which we term FDC-SP (FDC secreted protein). The FDC-SP gene lies on chromosome 4q13 adjacent to clusters of proline-rich salivary peptides and C-X-C chemokines. Human and mouse FDC-SP proteins are structurally unique and contain a conserved N-terminal charged region adjacent to the leader peptide. FDC-SP has a very restricted tissue distribution and is expressed by activated FDCs from tonsils and TNF-alpha-activated FDC-like cell lines, but not by B cell lines, primary germinal center B cells, or anti-CD40 plus IL-4-activated B cells. Strikingly, FDC-SP is highly expressed in germinal center light zone, a pattern consistent with expression by FDC. In addition, FDC-SP is expressed in leukocyte-infiltrated tonsil crypts and by LPS- or Staphylococcus aureus Cowan strain 1-activated leukocytes, suggesting that FDC-SP can also be produced in response to innate immunity signals. We provide evidence that FDC-SP is posttranslationally modified and secreted and can bind to the surface of B lymphoma cells, but not T lymphoma cells, consistent with a function as a secreted mediator acting upon B cells. Furthermore, we find that binding of FDC-SP to primary human B cells is markedly enhanced upon activation with the T-dependent activation signals such as anti-CD40 plus IL-4. Together our data identify FDC-SP as a unique secreted peptide with a distinctive expression pattern within the immune system and the ability to specifically bind to activated B cells.     

Localization of cap-binding protein in subcellular fractions of HeLa cells

The 26,000-M(r) cap-binding protein was analyzed by a cross-linking assay in cell fractions from uninfected and poliovirus-infected HeLa cells. Cap-binding protein was found in the postribosomal supernatant (S-200) and in the ribosomal salt wash. The cap-binding protein in the S-200 had a sedimentation coefficient of 5 to 7S and lacked the ability to restore translation in extracts of poliovirus-infected cells.     

Murine polo like kinase 1 gene is expressed in meiotic testicular germ cells and oocytes

To identify key molecules that regulate germ cell proliferation and differentiation, we have attempted to isolate protein kinase genes preferentially expressed in germ line cells. One such cDNA cloned from murine embryonic germ(EG) cells encodes a nonreceptor type serine/threonine kinase and is predominantly expressed in the testis, ovary, and spleen of adult mouse. The nucleotide sequence of the entire coding region shows that this clone, designated Plk1(polo like kinase 1), is identical with STPK13 previously cloned from murine erythroleukemia cells. The protein encoded by Plk1 is closely related to the product of Drosophila polo that plays a role in mitosis and meiosis. To define the role of Plk1 in germ cell development, we have examined its expression in murine gonads by in situ hybridization. Here we show that the PlK1 gene is specifically expressed in spermatocytes of diplotene and diakinesis stage, in secondary spermatocytes, and in round spermatids in testes. It is also expressed in growing oocytes and ovulated eggs. The pattern of expression of the Plk1 gene suggests that the gene product is involved in completion of meiotic division, and like the Drosophila polo protein, is a maternal factor active in embryos at the early cleavage stage. © 1995 Wiley-Liss, Inc.     

Both cell-surface and secreted CSF-1 expressed by tumor cells metastatic to bone can contribute to osteoclast activation

Tumors metastatic to the bone produce factors that cause massive bone resorption mediated by osteoclasts in the bone microenvironment. Colony stimulating factor (CSF-1) is strictly required for the formation and survival of active osteoclasts, and is frequently produced by tumor cells. Here we hypothesize that the CSF-1 made by tumor cells contributes to bone destruction in osteolytic bone metastases. We show that high level CSF-1 protected osteoclasts from suppressive effects of transforming growth factor β (TGF-β). r3T cells, a mouse mammary tumor cell line that forms osteolytic bone metastases, express abundant CSF-1 in vitro as both a secreted and a membrane-spanning cell-surface glycoprotein, and we show that both the secreted and the cell-surface form of CSF-1 made by r3T cells can support osteoclast formation in co-culture experiments in the presence of RankL. Mice with r3T bone metastases have elevated levels of both circulating and bone-associated CSF-1, and the majority of CSF-1 found in bone metastases is associated with the tumor cells. These results support the idea that tumor-cell produced CSF-1 contributes to osteoclast development and survival in bone metastasis.     

Binding of the CC-chemokine RANTES to syndecan-1 and syndecan-4 expressed on HeLa cells

It is believed that proteoglycans influence biological properties of chemokines. We show that the CC chemokine RANTES binds not only to high-affinity binding sites on CCR5-positive HeLa cells but also to low-affinity binding sites on HeLa cells expressing or lacking RANTES G protein-coupled receptors. Coimmunoprecipitation studies demonstrate that RANTES forms complexes with glycanated syndecan (SD)-1 and -4, in addition to CCR5 on the CCR5-positive HeLa cells. Moreover, confocal microscopy analysis shows the colocalization of RANTES with SD-1 and -4. Glycosaminoglycans removal from the cells by glycosaminidases treatment prevented RANTES binding to SD-1 and -4 and decreased RANTES binding to CCR5 on the CCR5-positive HeLa cells. Removal of glycosaminoglycans by glycosaminidases treatment of the complexes, RANTES/SD-1/SD-4/+/-CCR5, immobilized on beads, reversed SD-1 and -4 bindings. Therefore, RANTES bindings to SD-1 and -4 depend on glycosaminoglycans and facilitate RANTES interaction with CCR5. Extracting plasma membrane cholesterol abolished the coimmunoprecipitation of SD-1 with RANTES, suggesting that rafts are involved in RANTES association to SD-1. Confocal microscopy analysis as well as coimmunoprecipitation experiments show a RANTES-independent heteromeric complex on the CCR5-positive HeLa cells, SD-1, SD-4, and CCR5. This complex is likely a functional unit in which proteoglycans may modulate RANTES binding to CCR5.     

Ligand-dependent secretion of rat retinol-binding protein expressed in HeLa cells.

A minigene encoding rat retinol-binding protein (RBP) was transfected into HeLa cells, which do not express endogenous RBP, transthyretin, or cellular retinol-binding protein. The HeLa cells manufactured and secreted the transfected gene product, demonstrating that RBP-transthyretin assembly is not a requirement for the secretion of RBP. When HeLa cells were grown under vitamin A-deficient conditions, RBP accumulated in the endoplasmic reticulum. Both serum and retinol stimulated secretion of RBP in a concentration-dependent manner. The retinol-regulated secretion occurred also after protein synthesis had been blocked by cycloheximide. Addition of holo-RBP or retinal, but not retinoic acid, stimulated secretion of RBP. Thus, an in vitro model system that resembles the rat hepatocyte in vivo with regard to the known regulation of RBP secretion has been established in a human cell line of extrahepatic origin. It can be concluded that cellular retinol-binding protein is not required for the transfer of retinol to RBP and that the mechanism whereby retinol controls the intracellular transport of RBP is neither specific for tissues synthesizing RBP nor species-specific. To investigate the structural properties responsible for the endoplasmic reticulum retention of RBP in the absence of its ligand, a cDNA encoding chicken purpurin, a protein that is 50% identical to RBP and that binds retinol, was expressed in HeLa cells. In contrast to RBP, purpurin was not retained in vitamin A-deficient HeLa cells.