HPV16L1核浆运输的动力学过程

利用增强型绿色荧光蛋白(Enhancegreenflurenscentprotein,EGFP)标记不同的截短型HPV16L1蛋白(Humanpapillomavirustype16L1protein,HPV16L1),分析HPV16L1蛋白核定位信号(Nucleuslocationsignal,NLS)的作用。构建重组pFB-EGFP、pFB-EGFP-HPV16L1、pFB-EGFP-HPV16L1△NLS和pFB-EGFP-NLSHPV16L1p转移载体;在DH10Bac宿主菌内经Tn7转座子介导的同源重组后转染Sf-9细胞,获得重组Ac-EGFP、Ac-EGFP-HPV16L1、Ac-EGFP-HPV16L1△NLS和Ac-EGFP-NLSHPV16L1杆状病毒,感染Sf-9昆虫细胞表达相应截短型HPV16L1融合蛋白;利用荧光显微镜和激光共聚焦显微镜观察不同融合蛋白的荧光特性和核浆转运动力学过程。结果发现Ac-EGFP杆状病毒感染的Sf-9细胞内明亮的绿色荧光均匀分布;重组Ac-EGFP-HPV16L1和Ac-EGFP-NLSHPV16L1杆状病毒感染的Sf-9细胞,明亮的绿色荧光主要位于细胞核内;重组Ac-EGFP-HPV16L1△NLS杆状病毒感染的Sf-9细胞,绿色荧光局限于细胞浆内,细胞核内无绿色荧光。说明HPV16L1蛋白羧基端的23个氨基酸(GKRKATPTTSSTSTTAKRKKRKL)具有完全核定位作用,能引导HPV16L1蛋白和EGFP突破核膜屏障进入Sf-9细胞核内。     

Identification of two KH domain proteins in the alpha-globin mRNP stability complex.

Accumulation of globin mRNAs during erythroid differentiation is dependent on their extraordinary stability. The longevity of human alpha-globin mRNA is associated with a ribonucleoprotein complex (alpha-complex) formed on the 3' untranslated region (3'UTR). One or more of the proteins within this alpha-complex contain strong polycytosine [poly(C)] binding (alpha PCB) activity. In the present report we purify alpha PCB activity from human erythroid K562 cells. Although not able to bind the alpha-globin 3'UTR directly, alpha PCB activity is sufficient to complement alpha-complex formation in a cytosolic extract depleted of poly(C) binding activity. Peptide microsequencing demonstrates that alpha PCB activity contains two structurally related poly(C) binding proteins. These two proteins, alpha-complex protein (alpha CP)-1 and -2, have an overall structural identity of 80% and contain three repeats of the K homology (KH) domain which is found in a subset of RNA binding proteins. Epitope-tagged recombinant alpha CP-1 and alpha CP-2 expressed in cells are each incorporated into the alpha-complex. We conclude that alpha CP-1 and alpha CP-2, members of the KH domain RNA binding protein family, are involved in formation of a sequence-specific alpha-globin mRNP complex associated with alpha-globin mRNA stability. As such this represents the first example of a specific function for this class of proteins and suggests potential roles for other members of this protein family.     

Functional characterization of a testes-specific alpha-subunit isoform of the sodium/potassium adenosinetriphosphatase.

Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) alpha and beta subunits have been identified in mammals. The association of the various alpha and beta polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase alpha4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha4 and the Na pump beta1 subunit are coexpressed in the cells, Na, K-ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na(+)-dependent, K(+)-sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K(+). Furthermore, the activity of alpha4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase alpha4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta1 and beta3 subunits. In insect cells, the alpha4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha4beta1 and alpha4beta3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na(+), K(+), ATP, and ouabain. The enzymatic properties of alpha4beta1 and alpha4beta3 are, however, distinct from the other Na pump isozymes. A Na, K-ATPase activity with similar properties as the alpha4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the alpha4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.     

Human hemoglobin expression in Escherichia coli: importance of optimal codon usage.

The overexpression of a nonfusion product of human beta-globin in Escherichia coli from its cDNA sequence has been accomplished for the first time. Expression of beta-globin from its native cDNA required the use of the strong bacteriophage T7 promoter. In this system, beta-globin accumulated to approximately 10% of total E. coli proteins. alpha-Globin was not expressed in the T7 system using the native cDNA. For the expression of alpha-globin, synthetic genes containing optimal E. coli codons were constructed. Neither synthetic alpha- nor beta-globin gene alone was expressed from the lac or tac promoter. Globin expression was achieved when the two synthetic alpha- and beta-globin genes were combined as an operon downstream of the lac promoter. The two proteins combined intracellularly with endogenous heme, which was concomitantly overproduced to yield tetrameric hemoglobin as roughly 5-10% of total E. coli protein. Cloning the alpha- and beta-globin cDNAs in a construct identical with the lac promoter did not yield globin production, establishing the requirement for optimal codon usage. The recombinant beta-globin from the T7 expression system was purified and reconstituted in vitro with heme and native alpha chains. N-terminal analyses showed that the beta-globin produced in the T7 system and the tetrameric hemoglobin produced from the synthetic genes contained an additional beta 1 methionine residue. Two additional mutants, beta 1 Val----Met and beta 1 Val----Ala were produced using the T7 system. Functional and structural properties of the purified hemoglobins will be discussed in the following papers.     

Stable association of G proteins with beta 2AR is independent of the state of receptor activation.

beta 2-Adrenergic receptors expressed in Sf9 cells activate endogenous Gs and adenylyl cyclase [Mouillac B., Caron M., Bonin H., Dennis M. and Bouvier M. (1992) J. Biol. Chem. 267, 21733-21737]. However, high affinity agonist binding is not detectable under these conditions suggesting an improper stoichiometry between the receptor and the G protein and possibly the effector molecule as well. In this study we demonstrate that when beta 2-adrenergic receptors were co-expressed with various mammalian G protein subunits in Sf9 cells using recombinant baculoviruses signalling properties found in native receptor systems were reconstituted. For example, when beta 2AR was co-expressed with the Gs alpha subunit, maximal receptor-mediated adenylyl cyclase stimulation was greatly enhanced (60 +/- 9.0 versus 150 +/- 52 pmol cAMP/min/mg protein) and high affinity, GppNHp-sensitive, agonist binding was detected. When G beta gamma subunits were co-expressed with Gs alpha and the beta 2AR, receptor-stimulated GTPase activity was also demonstrated, in contrast to when the receptor was expressed alone, and this activity was higher than when beta 2AR was co-expressed with Gs alpha alone. Other properties of the receptor, including receptor desensitization and response to inverse agonists were unaltered. Using antisera against an epitope-tagged beta 2AR, both Gs alpha and beta gamma subunits could be co-immunoprecipitated with the beta 2AR under conditions where subunit dissociation would be expected given current models of G protein function. A desensitization-defective beta 2AR (S261, 262, 345, 346A) and a mutant which is constitutively desensitized (C341G) could also co-immunoprecipitate G protein subunits. These results will be discussed in terms of a revised view of G protein-mediated signalling which may help address issues of specificity in receptor/G protein coupling.     

Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.

The extracellular domain of the 55-kDa TNF receptor (rsTNFR beta) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr- cells. A chimeric fusion protein (rsTNFR beta-h gamma 3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG C gamma 3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNF alpha- or protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFR beta was found to bind TNF alpha with high affinity comparable with the membrane-bound full-length receptor. The affinity for TNF beta was slightly impaired. However, the bivalent rsTNFR beta-h gamma 3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFR beta reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFR beta.TNF alpha and rsTNFR beta.TNF beta complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFR beta molecules bound to one TNF alpha or TNF beta trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFR beta and its TNF alpha and TNF beta complexes. Multiple receptor binding sites on TNF alpha trimers could also be demonstrated by a TNF alpha-induced agglutination of Latex beads coated with the rsTNFR beta-h gamma 3 fusion protein. Both rsTNFR beta and rsTNFR beta-h gamma 3 were found to inhibit binding of TNF alpha and TNF beta to native 55- and 75-kDa TNF receptors and to prevent TNF alpha and TNF beta bioactivity in a cellular cytotoxicity assay. Concentrations of rsTNFR beta-h gamma 3 equimolar to TNF alpha were sufficient to neutralize TNF activity almost completely, whereas a 10-100-fold excess of rsTNFR beta was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.     

Expression of alpha- and beta-globin genes occurs within different nuclear domains in haemopoietic cells

The alpha- and beta-globin gene clusters have been extensively studied. Regulation of these genes ensures that proteins derived from both loci are produced in balanced amounts, and that expression is tissue-restricted and specific to developmental stages. Here we compare the subnuclear location of the endogenous alpha- and beta-globin loci in primary human cells in which the genes are either actively expressed or silent. In erythroblasts, the alpha- and beta-globin genes are localized in areas of the nucleus that are discrete from alpha-satellite-rich constitutive heterochromatin. However, in cycling lymphocytes, which do not express globin genes, the distribution of alpha- and beta-globin genes was markedly different. beta-globin loci, in common with several inactive genes studied here (human c-fms and SOX-1) and previously (mouse lambda5, CD4, CD8alpha, RAGs, TdT and Sox-1), were associated with pericentric heterochromatin in a high proportion of cycling lymphocytes. In contrast, alpha-globin genes were not associated with centromeric heterochromatin in the nucleus of normal human lymphocytes, in lymphocytes from patients with alpha-thalassaemia lacking the regulatory HS-40 element or entire upstream region of the alpha-globin locus, or in mouse erythroblasts and lymphocytes derived from human alpha-globin transgenic mice. These data show that the normal regulated expression of alpha- and beta-globin gene clusters occurs in different nuclear environments in primary haemopoietic cells.     

The alpha and beta subunits of the Na,K-ATPase can assemble at the plasma membrane into functional enzyme

Synthesis and assembly of most oligomeric plasma membrane proteins occurs in the ER. However, the role the ER plays in oligomerization is unknown. We have previously demonstrated that unassociated alpha and beta subunits of the Na,K-ATPase are targeted to the plasma membrane when individually expressed in baculovirus-infected Sf-9 cells. This unique property allows us to determine if assembly of these two polypeptides is restricted to the ER, or if it can also occur at the plasma membrane. To investigate the assembly of the Na,K-ATPase we have taken advantage of the ability of baculovirus-infected cells to fuse. Lowering the extracellular pH of the infected cells triggers an endogenously expressed viral protein to initiate plasma membrane fusion. When individual Sf-9 cells expressing either the Na,K-ATPase alpha or beta subunits are plated together and subjected to a mild acidic shock, they form large syncytia. In the newly continuous plasma membrane the separate alpha and beta polypeptides associate and assemble into functional Na,K-ATPase molecules. However, a hybrid ATPase molecule consisting of a Na,K-ATPase alpha subunit and a H,K- ATPase beta subunit, which efficiently assembles in the ER of coinfected cells, does not assemble at the plasma membrane of fused cells. When cells expressing the Na,K-ATPase alpha subunit are fused to cells coexpressing the Na,K-ATPase beta subunit and the H,K-ATPase beta subunit, the Na,K-ATPase alpha subunit selectively assembles with the Na,K-ATPase beta subunit. However, when cells are coinfected and expressing all three polypeptides, the Na,K-ATPase alpha subunit assembles with both beta subunits in the ER, in what appears to be a random fashion. These experiments demonstrate that assembly between some polypeptides is restricted to the ER, and suggests that the ability of the Na,K-ATPase alpha and beta subunits to leave the ER and assemble at the plasma membrane may represent a novel mechanism of regulation of activity.     

Secretion and purification of recombinant beta1-4 galactosyltransferase from insect cells using pFmel-protA, a novel transposition-based baculovirus transfer vector

The palette of transfer vectors available for generation of recombinant baculoviruses based on transposition-mediated recombination has been enlarged by constructing the pFmel-protA vector. The pFmel-protA plasmid includes the honeybee melittin secretion signal and a Staphylococcus aureus protein A fusion protein tag, which allows the secretion and purification of recombinant proteins. Using this system, the human beta1-4 galactosyltransferase-I protein was expressed in Sf9 insect cells at a level ranging from 22 to 28 U (4.8 to 6.0 mg)/L. The protein A tag enabled a simple monitoring of recombinant protein expression by enzyme-linked immunosorbent assay and Western blotting. Single step purification was achieved by immunoglobulin G affinity chromatography achieving a recovery yield of 28% and a specific activity of 1.9 U per mg of recombinant protein.     

Cis-acting sequences regulating expression of the human alpha-globin cluster lie within constitutively open chromatin.

Current models suggest that tissue-specific genes are arranged in discrete, independently controlled segments of chromatin referred to as regulatory domains. Transition from a closed to open chromatin structure may be an important step in the regulation of gene expression. To determine whether the human alpha-globin cluster, like the beta-globin cluster, lies within a discrete, erythroid-specific domain, we have examined the long-range genomic organization and chromatin structure around this region. The alpha genes lie adjacent to at least four widely expressed genes. The major alpha-globin regulatory element lies 40 kb away from the cluster within an intron of one of these genes. Therefore, unlike the beta cluster, cis-acting sequences controlling alpha gene expression are dispersed within a region of chromatin that is open in both erythroid and nonerythroid cells. This implies a difference in the hierarchical control of alpha- and beta-globin expression.     

Heme-regulated eIF2alpha kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency.

Although the physiological role of tissue-specific translational control of gene expression in mammals has long been suspected on the basis of biochemical studies, direct evidence has been lacking. Here, we report on the targeted disruption of the gene encoding the heme-regulated eIF2alpha kinase (HRI) in mice. We establish that HRI, which is expressed predominantly in erythroid cells, regulates the synthesis of both alpha- and beta-globins in red blood cell (RBC) precursors by inhibiting the general translation initiation factor eIF2. This inhibition occurs when the intracellular concentration of heme declines, thereby preventing the synthesis of globin peptides in excess of heme. In iron-deficient HRI(-/-) mice, globins devoid of heme aggregated within the RBC and its precursors, resulting in a hyperchromic, normocytic anemia with decreased RBC counts, compensatory erythroid hyperplasia and accelerated apoptosis in bone marrow and spleen. Thus, HRI is a physiological regulator of gene expression and cell survival in the erythroid lineage.     

Expression of functional soluble human alpha-globin chains of hemoglobin in bacteria

Individual, soluble human alpha-globin chains were expressed in bacteria with exogenous heme and methionine aminopeptidase. The yields of soluble alpha chains in bacteria were comparable to those of recombinant non-alpha chains expressed under the same conditions. Molecular mass and gel-filtration properties of purified recombinant alpha chains were the same as those of authentic human alpha chains. Biochemical and biophysical properties of isolated alpha chains were identical to those of native human alpha chains as assessed by UV/vis, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy which contrasts with previous results of refolded precipitated alpha chains made in the presence of heme in vitro (M. T. Sanna et al., J. Biol. Chem. 272, 3478-3486, 1997). Mixtures of purified, soluble recombinant alpha-globin and native beta-globin chains formed heterotetramers in vitro, and oxygen- and CO-binding properties as well as the heme environment of the assembled tetramers were experimentally indistinguishable from those of native human Hb A. UV/vis, CD, and NMR spectra of assembled Hb A were also the same as those of human Hb A. These results indicate that individual expressed alpha chains are stable in bacteria and fold properly in vivo and that they then can assemble with free beta chains to form hemoglobin heterotetramers in vivo as well as in vitro.     

Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli.

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.     

Electrophoretic analysis of glycoprotein glycans produced by lepidopteran insect cells infected with an immediate early recombinant baculovirus encoding mammalian β1,4-galactosyltransferase

Glycosylation, the most extensive co- and post-translational modification of eukaryotic cells, can significantly affect biological activity and is particularly important for recombinant glycoproteins in human therapeutic applications. The baculovirus-insect cell expression system is a popular tool for the expression of heterologous proteins and has an excellent record of producing high levels of biologically active eukaryotic proteins. Insect cells are capable of glycosylation, but their N-glycosylation pathway is truncated in comparison with the pathway of mammalian cells. A previous study demonstrated that an immediate early recombinant baculovirus could be used to extend the insect cell N-glycosylation pathway by contributing bovine -1,4 galactosyltransferase (GalT) immediately after infection. Lectin blotting assays indicated that this ectopically expressed enzyme could transfer galactose to an N-linked glycan on a foreign glycoprotein expressed later in infection. In the current study, glycans were isolated from total Sf-9 cell glycoproteins after infection with the immediate early recombinant baculovirus encoding GalT, fluorescently conjugated and analyzed by electrophoresis in combination with exoglycosidase digestion. These direct analyses clearly demonstrated that Sf-9 cells infected with this recombinant baculovirus can synthesize galactosylated N-linked glycans.     

Characterization and expression of embryonic and adult globins of the teleost Oryzias latipes (medaka)

Using the teleost Oryzias latipes (medaka), we isolated three embryonic globin cDNAs (em.alpha-0, em.alpha-1, and em.beta-1) from the embryos 5 days after fertilization (at 30 degrees C) and two adult globin cDNAs (ad.alpha-1 and ad.beta-1) from the kidney of the fully-grown adult fish, and predicted their amino acid sequences. Molecular phylogenetic analysis showed that the embryonic globins were highly homologous in amino acid sequence to the embryonic globins previously identified in rainbow trout and zebrafish, and that they formed a monophyletic group among the teleostean globin molecules. They were clearly discriminated from the adult globin of the medaka. RT-PCR analysis showed that the embryonic globin mRNAs were intensely expressed in stage 30 and 38 embryos and in young fish 30 days after hatching. The level of expression decreased drastically after the young fish stage, and was low in fully-grown adult fish. The adult alpha globin mRNA ad.alpha-1 was scarcely expressed in the embryos, and the level of expression gradually increased in young to fully-grown adult fish. Unexpectedly, the adult beta globin mRNA ad.beta-1 was expressed throughout life, from the early embryonic stage to the fully-grown adult stage. This expression profile was quite different from that of the rainbow trout previously investigated. Some globins of the medaka were expressed both in primitive hematopoiesis and in definitive hematopoiesis.     

Culture in the rotating-wall vessel affects recombinant protein production capability of two insect cell lines in different manners

Summary The production of recombinant secreted alkaline phosphatase protein in virally infected insect cells was studied in shaker flask and high aspect rotating-wall vessel (HARV) culture. Two commonly used cell lines, Spodoptera frugiperda Sf-9 (Sf-9) and a nonaggregating isolate of the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) cell line, Trichoplusia ni Tn-5B1-4-NA (TN-5B1-4-NA), were used and monitored for 120-h postinfection. Different responses to culture in the HARV were seen in the two cell lines. While the Sf-9 cell line was able to produce slightly greater amounts of recombinant protein in the HARV than in shaker flask controls, the Tn-5B1-4-NA cell line produced significantly lesser amounts in the HARV than in the shaker flasks. Both cell lines exhibited longer life spans and longer periods of protein production in HARV culture than in shaker flask culture, presumably due to lower levels of shear encountered in the HARV. The important difference was in the protein production rate responses of the two cell lines. While the protein production rates of Sf-9 cells were comparable in both HARV and shaker flask cultures, the protein production rates of Tn-5B1-4-NA cells were much lower in HARV culture than in shaker flask cultures. The conclusion is drawn that cell line-specific adaptation to the HARV strongly influences recombinant protein production.