Development of a new epitope tag recognized by a monoclonal antibody to Rickettsia typhi

The epitope recognized by a mouse monoclonal antibody (MAb) to the crystalline surface layer protein of Rickettsia typhi, SRT10, was mapped to 10 amino acid residues (SRTag TFIGAIATDT). The oligonucleotide sequence covering the epitope recognized by SRT10 was inserted into a mammalian expression vector together with multiple cloning sites. When the SRTag was fused in frame to the coding region of the NCC27/CLIC1 gene and expressed in mammalian cells, the MAb SRT10 could detect the tagged protein by immunoblotting, immunocytochemistry, and immunoprecipitation. In addition to the SRT-NCC27/CLIC1, SRT10 could detect N-terminal-tagged MEF2D and C-terminal-tagged CD4 by immunocytochemistry. We suggest that this specific recognition of the SRTag by SRT10 is generally applicable to cellular and molecular biology research that requires the expression and detection of fusion proteins.     

Activities of the EM10 protein from Echinococcus multilocularis in cultured mammalian cells demonstrate functional relationships to ERM family members

The ezrin-radixin-moesin (ERM) homolog EM10 is expressed by the larval stage of the parasite E. multilocularis and shows 46.9% overall identity in the primary structure with human ezrin. To determine whether EM10 has similar activities to ERM proteins, we investigated properties of the protein expressed in mammalian cells. In particular, we transiently expressed haemagglutinin-tagged (HA-tagged) versions of the full-length EM10 as well as the amino- and the carboxy-terminal halves of EM10 in HtTA-1 cells. In addition we stably transfected NIH-3T3 cells with untagged full-length EM10. The data demonstrate that EM10 polypeptides behave like their corresponding portions of radixin when transiently expressed in mammalian cells. The full-length and amino-terminal EM10 polypeptides were localized to cortical structures. Cells expressing the carboxy-terminal polypeptide of EM10 showed long actin-filled protrusions. Cells expressing full-length EM10 showed a reduction in endogenous moesin-staining at cortical structures. In stably transfected NIH-3T3 cells EM10 was not crisply localized but rather was diffuse throughout the cytoplasm. These cells showed a conspicuous loss of stress-fibers, a phenotype that was not seen in analogous experiments with ERM proteins. The results demonstrate both similarities and differences between the functional properties of EM10 and ERM proteins expressed in vertebrate cells.     

Transfer of monoclonal antibodies into mammalian cells by electroporation

A simple rapid and reproducible procedure for transferring monoclonal antibodies into mammalian cells by electroporation is described. Two functionally different monoclonal antibodies (Mab 3F3 and Mab 2B4) specific for asparagine synthetase (EC 6.3.1.1) were used for electroporation into HeLa, HT-5, and L5178Y D10/R (L-asparaginase-resistant) cells. The conditions were optimized so that the viability of the electroporated cells was very high (80-90%), and 90% of the viable cells had antibody incorporated. Electropermeabilized cells were structurally intact, and the high voltage electric pulse had no inhibitory effect on overall cellular DNA and protein synthesis. Incorporated immunoglobulins showed unaltered structural integrity and were functionally active. L5178Y D10/R cells incorporated with an antibody (Mab 3F3) known to be a potent inhibitor of tumor asparagine synthetase showed increased dependence on an exogenous source of asparagine in the culture medium, while the growth of cells incorporated with a control (noninhibitory) antibody (Mab 2B4) remained unaffected. These studies demonstrate that electroporation can be employed successfully for large scale transfer of antibodies into cultured mammalian cells for the study of cellular metabolism.     

Characterization of a human cervical carcinoma-associated antigen by lectin binding and immuno-electron microscopy.

The specific binding and nature of the epitope recognized by monoclonal antibody (Mab) 1H10, which binds an antigen expressed on human cervical tumors, was characterized by enzyme digestion, lectin competition assay and immuno-electron microscopy. Membrane homogenates of CaSki cervical carcinoma cells were digested with various enzymes, then analysed by SDS-PAGE and immunoblotting. Cells grown on coverslips were treated with various enzymes and in situ binding of Mab 1H10 to cells was analysed by electron microscopy. The ability of lectin-conjugates to block Mab 1H10 binding to CaSki cells was also examined. Treatment of samples with sodium periodate abrogated antigen recognition by Mab 1H10. Neuraminidase and hyaluronidase digestion decreased but did not eliminate Mab 1H10 binding to cells in situ. Chondroitinase ABC digestion, in contrast, removed Mab 1H10 binding sites both in vitro and in situ. Trypsin and chymotrypsin digestion of cell membrane homogenates decreased the molecular weight of the Mab 1H10 antigen but did not decrease the binding intensity. Wheat germ agglutinin (WGA) strongly bound to CaSki cells and partially blocked Mab 1H10 binding, indicating that the antigen contains N-acetyl-galactosamine residues at or near the epitope recognized by Mab 1H10. Ricinus communis agglutinin (RCA) exhibited a similar binding pattern to WGA. However, concanavalin A bound only weakly to CaSki cells and was ineffective at blocking Mab 1H10 binding. The tumor-associated antigen recognized by Mab 1H10 is concluded to be a chondroitin sulphate glycoprotein or proteoglycan rather than a mucopolysaccharide or lipoprotein.     

Characterization of a human cervical carcinoma-associated antigen by lectin binding and immuno-electron microscopy

Summary The specific binding and nature of the epitope recognized by monoclonal antibody (Mab) 1H10, which binds an antigen expressed on human cervical tumors, was characterized by enzyme digestion, lectin competition assay and immuno-electron microscopy. Membrane homogenates of CaSki cervical carcinoma cells were digested with various enzymes, then analysed by SDS-PAGE and immunoblotting. Cells grown on coverslips were treated with various enzymes and in situ binding of Mab 1H10 to cells was analysed by electron microscopy. The ability of lectin-conjugates to block Mab 1H10 binding to CaSki cells was also examined. Treatment of samples with sodium periodate abrogated antigen recognition by Mab 1H10. Neuraminidase and hyaluronidase digestion decreased but did not eliminate Mab 1H10 binding to cells in situ. Chondroitinase ABC digestion, in contrast, removed Mab 1H10 binding sites both in vitro and in situ. Trypsin and chymotrypsin digestion of cell membrane homogenates decreased the molecular weight of the Mab 1H10 antigen but did not decrease the binding intensity. Wheat germ agglutinin (WGA) strongly bound to CaSki cells and partially blocked Mab 1H10 binding, indicating that the antigen contains N-acetyl-galactosamine residues at or near the epitope recognized by Mab 1H10. Ricinus communis agglutinin (RCA) exhibited a similar binding pattern to WGA. However, concanavalin A bound only weakly to CaSki cells and was ineffective at blocking Mab 1H10 binding. The tumor-associated antigen recognized by Mab 1H10 is concluded to be a chondroitin sulphate glycoprotein or proteoglycan rather than a mucopolysaccharide or lipoprotein.     

Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells

Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines.     

GATA DNA-binding protein expressed in mouse I-10 Leydig testicular tumor cells

A nuclear extract of the mouse I-10 Leydig tumor cell line was analyzed by gel mobility shift assay with a combination of antibodies for various mammalian GATA proteins. Antibodies for GATA-4 caused a super-shift of the DNA-protein complex, which is formed through GATA-4 binding to an oligonucleotide with a typical GATA motif, while ones for GATA-1, GATA-2, GATA-3, and GATA-6 did not. These results indicated that I-10 cells express GATA-4 protein. Western blotting analysis of cellular proteins also demonstrated the presence of GATA-4 protein, the size of which corresponds to that of the rat orthologous protein transiently expressed in Cos-1 cells. A significant level of GATA-4 expression in I-10 cells would be advantageous for studying the roles of this protein, especially in view of gonadal function. We further examined the binding site preference of GATA-4 expressed in I-10 cells. GATA-4 showed broad sequence specificity similar to GATA-6, the order of binding core site preference being GATA > GATT > GATC, and adenine was favored on both sides of the core for strong binding. Thus the conserved zinc finger domain of GATA proteins is suggested to contribute to the binding sequence preference. GATA-4 expressed in I-10 cells was not susceptible to proteolysis coupled with cAMP signaling.     

Phosphorylation of Maize Eukaryotic Translation Initiation Factor 5A (eIF5A) by Casein Kinase 2: IDENTIFICATION OF PHOSPHORYLATED RESIDUE AND INFLUENCE ON INTRACELLULAR LOCALIZATION OF eIF5A*

Maize eukaryotic translation initiation factor 5A (ZmeIF5A) co-purifies with the catalytic α subunit of protein kinase CK2 and is phosphorylated by this enzyme. Phosphorylated ZmeIF5A was also identified after separation of maize leaf proteins by two-dimensional electrophoresis. Multiple sequence alignment of eIF5A proteins showed that in monocots, in contrast to other eukaryotes, there are two serine/threonine residues that could potentially be phosphorylated by CK2. To identify the phosphorylation site(s) of ZmeIF5A, the serine residues potentially phosphorylated by CK2 were mutated. ZmeIF5A and its mutated variants S2A and S4A were expressed in Escherichia coli and purified. Of these recombinant proteins, only ZmeIF5A-S2A was not phosphorylated by maize CK2. Also, Arabidopsis thaliana and Saccharomyces cerevisiae eIF5A-S2A mutants were not phosphorylated despite effective phosphorylation of wild-type variants. A newly developed method exploiting the specificity of thrombin cleavage was used to confirm that Ser² in ZmeIF5A is indeed phosphorylated. To find a role of the Ser² phosphorylation, ZmeIF5A and its variants mutated at Ser² (S2A and S2D) were transiently expressed in maize protoplasts. The expressed fluorescence labeled proteins were visualized by confocal microscopy. Although wild-type ZmeIF5A and its S2A variant were distributed evenly between the nucleus and cytoplasm, the variant with Ser² replaced by aspartic acid, which mimics a phosphorylated serine, was sequestered in the nucleus. These results suggests that phosphorylation of Ser² plays a role in regulation of nucleocytoplasmic shuttling of eIF5A in plant cells.     

Nuclear export of S6K1 II is regulated by protein kinase CK2 phosphorylation at Ser-17

Ribosomal S6 kinases (S6Ks) are principal players in the regulation of cell growth and energy metabolism. Signaling via phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways mediates the activation of S6K in response to various mitogenic stimuli. The family of S6Ks consists of two forms, S6K1 and -2, that have cytoplasmic and nuclear splicing variants, S6K1 II and S6K1 I, respectively. Nuclear-cytoplasmic shuttling of both isoforms induced by mitogenic stimuli has been reported recently. Here we present the identification of protein kinase CK2 (CK2) as a novel binding and regulatory partner for S6K1 II. The interaction between S6K1 II and CK2beta regulatory subunit was initially identified in a yeast two-hybrid screen and further confirmed by co-immunoprecipitation of transiently expressed and endogenous proteins. The interaction between S6K1 II and CK2 was found to occur in serum-starved and serum-stimulated cells. In addition, we found that S6K1 II is a substrate for CK2. The localization of the CK2 phosphorylation site was narrowed down to Ser-17 in S6K1 II. Mutational analysis and the use of phosphospecific antibody indicate that Ser-17 is a major in vitro and in vivo phosphorylation site for CK2. Functional studies reveal that, in contrast to the wild type kinase, the phosphorylation-mimicking mutant of S6K1 II (S17E) retains its cytoplasmic localization in serum-stimulated cells. Treatment of cells with the nuclear export inhibitor leptomycin B revealed that the S17E mutant accumulates in the nucleus to the same extent as S6K1 II wild type. These results indicate that nuclear import of the S17E mutant is not affected, although the export is significantly enhanced. We also provide evidence that nuclear export of S6K1 is mediated by a CRM1-dependent mechanism. Taken together, this study establishes a functional link between S6K1 II and CK2 signaling, which involves the regulation of S6K1 II nuclear export by CK2-mediated phosphorylation of Ser-17.     

Ectopic synthesis of epidermal cytokeratins in pancreatic islet cells of transgenic mice interferes with cytoskeletal order and insulin production

The members of the multigene family of intermediate filament (IF) proteins are expressed in various combinations and amounts that are specific for a given pathway or state of differentiation. Previous experiments in which the cell type-specific IF cytoskeleton was altered by introducing foreign IF proteins into cultured cells or certain tissues of transgenic animals have shown a remarkable tolerance, without detectable interference with cell functions. To examine the importance of the cell type-specific cytokeratin (CK) IF pattern, we have studied the ectopic expression of CK genes in different epithelia of transgenic mice. Here we report changes observed in the beta cells of pancreatic islets expressing the genes for human epidermal CKs 1 and/or 10 brought under control of the rat insulin promoter. Both genes were efficiently expressed, resulting in the appearance of numerous and massive bundles of aggregated IFs, resembling those of epidermal keratinocytes. While the synthesis of epidermal CK 10 was readily accommodated and compatible with cell function, mice expressing CK 1 in their beta cells, alone or in combination with CK 10, developed a special form of diabetes characterized by a drastic reduction of insulin-secretory vesicles and of insulin-and CK 1-producing cells. In many CK 1-producing cells, accumulations of fibrous or granular material containing CK 1 were also seen in the nucleus. This demonstration of functional importance of the specific CK-complement in an epithelial cell indicates a contribution of cell type-specific factors to cytoplasmic IF compartmentalization and that the specific CK complement can be crucial for functions and longevity of a given kind of epithelium.     

Expression of a genomic clone encoding a brain potassium channel in mammalian cells using lipofection

A genomic clone encoding a mouse brain K⁺ channel (MBK1) was isolated, characterized and expressed in COS cells using the lipofection technique. Transfected COS cells expressed voltage-dependent K⁺ currents that activated within 20 ms at 0 mV and showed less than 10% inactivation during 250 ms depolarizing pulses at 60 mV. Expressed K⁺ currents were reversibly blocked by 4-aminopyridine and tetraethylammonium, and were moderately sensitive to dendrotoxin, but insensitive to charybdotoxin. Thus MBK1, expressed transiently in a mammalian cell line, exhibits features characteristic of non-inactivating K⁺ channels with a conspicuous insensitivity to charybdotoxin. Lipofection is, therefore, a valuable strategy for expression of channel proteins in mammalian cells.Abbreviations 4-AP 4 aminopyridine - TEA tetraethylammonium - CTX charybdotoxin - DTX dendrotoxin - V applied voltage - V_rev reversal potential - I current - G conductance - MBK1 mouse brain potassium channel 1 - TES N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid Correspondence to: M. Montal.     

应用蛋白质组学方法初步筛选与鼻咽癌放射敏感相关的蛋白

目的:通过筛选放射敏感性不同的鼻咽癌细胞中差异表达蛋白,以发现与鼻咽癌放射敏感相关的蛋白。方法:放射处理并结合流式细胞术检测及比较5-8F和6-10B细胞的放射敏感性。提取细胞总蛋白,进行双向凝胶电泳、MALDI-TOF肽质指纹图分析、质谱数据的蛋白质库搜寻鉴定。应用Western Blot检测细胞中蛋白质表达。应用免疫组织化学方法检测鼻咽癌组织中相关蛋白的表达。结果:双向凝胶电泳后对胶上的部分分辨较好的差异蛋白质点进行肽质谱指纹图分析和鉴定,在两种细胞中差异表达最为显著的蛋白质有9个。Western Blot证实CK19和P73在5-8F和6-10B表达与蛋白质组结果一致。P73在鼻咽癌放射敏感组和不敏感组中的表达阳性率分别为90%、57.5%,存在显著性差异。结论:放射敏感性不同的鼻咽癌细胞中存在一些差异表达蛋白,这些蛋白可能与鼻咽癌放射敏感性有关,其中P73可能成为放射敏感性预测的侯选标志物。     

Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid

We report a method of photo-cross-linking proteins in mammalian cells, which is based on site-specific incorporation of a photoreactive amino acid, p-benzoyl-L-phenylalanine (pBpa), through the use of an expanded genetic code. To analyze the cell signaling interactions involving the adaptor protein Grb2, pBpa was incorporated in its Src homology 2 (SH2) domain. The human GRB2 gene with an amber codon was introduced into Chinese hamster ovary (CHO) cells, together with the genes for the Bacillus stearothermophilus suppressor tRNA(Tyr) and a pBpa-specific variant of Escherichia coli tyrosyl-tRNA synthetase (TyrRS). The Grb2 variant with pBpa in the amber position was synthesized when pBpa was included in the growth medium. Upon exposure of cells to 365-nm light, protein variants containing pBpa in the positions proximal to the ligand-binding pocket were cross-linked with the transiently expressed epidermal growth factor (EGF) receptor in the presence of an EGF stimulus. Cross-linked complexes with endogenous proteins were also detected. In vivo photo-cross-linking with pBpa incorporated in proteins will be useful for studying protein-protein interactions in mammalian cells.     

On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio of heavy chain (HC) and light chain (LC) polypeptides. Accordingly, most recombinant Mab expression systems utilize an equimolar ratio of heavy chain (hc) to light chain (lc) genes encoded on either one or two plasmids. However, there is no evidence to suggest that this gene ratio is optimal for stable or transient production of recombinant Mab. In this study we have determined the optimal ratio of hc:lc genes for production of a recombinant IgG4 Mab, cB72.3, by Chinese hamster ovary (CHO) cells using both empirical and mathematical modeling approaches. Polyethyleneimine-mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmids yielded an optimal Mab titer at a hc:lc gene ratio of 3:2; a conclusion confirmed by separate mathematical modeling of the Mab folding and assembly process using transient expression data. On the basis of this information, we hypothesized that utilization of hc genes at low hc:lc gene ratios is more efficient. To confirm this, cB72.3 Mab was transiently produced by CHO cells at constant hc and varying lc gene dose. Under these conditions, Mab yield was increased with a concomitant increase in lc gene dose. To determine if the above findings also apply to stably transfected CHO cells producing recombinant Mab, we compared the intra- and extracellular ratios of HC and LC polypeptides for three GS-CHO cells lines transfected with a 1:1 ratio of hc:lc genes and selected for stable expression of the same recombinant Mab, cB72.3. Intra- and extracellular HC:LC polypeptide ratios ranged from 1:2 to 1:5, less than that observed on transient expression of the same Mab in parental CHO cells using the same vector. In conclusion, our data suggest that the optimal ratio of hc:lc genes used for transient and stable expression of Mab differ. In the case of the latter, we infer that optimal Mab production by stably transfected cells represents a compromise between HC abundance limiting productivity and the requirement for excess LC to render Mab folding and assembly more efficient.     

Monoclonal antibodies ICO-20 against human thymocyte antigen T10

Monoclonal antibodies (Mab) Ig G2a isotypes reacting in indirect immunofluorescence assay with 68.7 +/- 4.1% of thymocytes, 7% of T-cells and not determining the antigen on other blood cells were obtained. Mab ICO-20 reacted in complement-dependent cytotoxic test. The antigen was expressed on colony-forming cells of granulocyte-macrophage row. Mab ICO-20 reaction with 100% of thymocytes was defined by flow cytometry. Antigen molecular mass is 45000 Dalton. The antigen was expressed on blast cells of patients with ALL and AML. Mab ICO-20 reaction was more more often with T-cell ALL.     

登革2型病毒衣壳蛋白在哺乳动物细胞中的表达与鉴定

从构建的重组质粒pLEX—C中高保真PCR获得编码登革2型病毒43株C基／E／（D2C）DNA片段,通过基因重组的方法将其克隆入真核表达载体pcDNA6／V5-His获得了重组真核表达载体pc／D2C。经电穿孔的方法转染BHK21细胞后,分别通过RT—PCR、免疫荧光和western印迹鉴定表达的蛋白。结果重组蛋白在BHK21细胞中获得表达,表达的蛋自主要存在于胞浆中,并具有较好的抗原性,能够被抗登革病毒衣壳蛋白单克隆抗体特异识别。此研究为深入了解登革病毒衣壳蛋白在病毒复制及组装过程中的生物学功能奠定了基础。     

Evaluation of the role of the endoplasmic reticulum-Golgi transit in the biogenesis of peroxisomal membrane proteins in wild type and peroxisome biogenesis mutant CHO cells

Peroxisomes are thought to be formed by division of pre-existing peroxisomes after the import of newly synthesized proteins. However, it has been recently suggested that the endoplasmic reticulum (ER) provides an alternative de novo mechanism for peroxisome biogenesis in some cells. To test a possible role of the ER-Golgi transit in peroxisome biogenesis in mammalian cells, we evaluated the biogenesis of three peroxisomal membrane proteins (PMPs): ALDRP (adrenoleukodystrophy related protein), PMP70 and Pex3p in CHO cells. We constructed chimeric genes encoding these PMPs and green fluorescent protein (GFP), and transiently transfected them to wild type and mutant CHO cells, in which normal peroxisomes were replaced by peroxisomal membrane ghosts. The expressed proteins were targeted to peroxisomes and peroxisomal ghosts correctly in the presence or absence of Brefeldin A (BFA), a drug known to block the ER-Golgi transit. Furthermore, low temperature did not disturb the targeting of Pex3p-GFP to peroxisomes. We also constructed two chimeric proteins of PMPs containing an ER retention signal "DEKKMP": GFP-ALDRP-DEKKMP and myc- Pex3p-DEKKMP. These proteins were mostly targeted to peroxisomes. No colocalization with an ER maker was found. These results suggest that the classical ER-Golgi pathway does not play a major role in the biogenesis of mammalian PMPs.     

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10‐R98S). Our data support that RPL10‐R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock‐in of RPL10‐R98S was associated with a 1.7‐ to 2.5‐fold increased production of four transiently expressed recombinant proteins and 1.7‐fold for one out of two stably expressed proteins. In CHO‐S cells, eGFP reached a 2‐fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10‐R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10‐R98S phenotypes can maximize its exploitability for the mammalian protein production industry.