In vitro expression and secretion of functional mammalian intrinsic factor using recombinant baculovirus.

Intrinsic factor was produced at levels of 1-2 mg per 1 (0.25 micrograms per 10(6) cells) by growth of recombinant baculovirus-infected Sf9 cells in spinner culture. The recombinant IF showed a binding affinity for cobalamin (2.6.10(-10) M) and for the intrinsic factor-cobalamin receptor (3.5.10(-10) M) nearly identical with native IF. Purification of the recombinant intrinsic factor could be accomplished by affinity chromatography, but final purification by gel chromatography (FPLC) was necessary to separate intrinsic factor from a 62 kDa protein secreted from uninfected Sf9 cells. This protein binds selectively to the cobalamin-Sepharose column, but demonstrates no cobalamin binding activity after elution. Microgram quantities of radiolabelled protein could be produced for metabolic and autoradiographic studies. The stability of intrinsic factor to pancreatic proteinases was nearly identical with human gastric intrinsic factor, both native and recombinant as produced in mammalian cells. Glycosylation of the intrinsic factor was demonstrated by lectin binding to the recombinant protein separated on SDS-PAGE, and by a shift in apparent molecular mass from 47 kDa to 43 kDa following treatment of Sf9 cells with tunicamycin. Most of the recombinant IF was produced by Sf9 cells in the first 48 h post infection.     

Production of a urokinase plasminogen activator-IgG fusion protein (uPA-IgG) in the baculovirus expression system

Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPAIgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 μg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 μg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA.     

A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells

Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.     

人视黄醇结合蛋白4在杆状病毒系统中的表达及其多克隆抗体制备

旨在利用杆状病毒系统表达、制备人视黄醇结合蛋白(RBP4)并检测其免疫原性。将人RBP4基因片段及信号肽SS64片段亚克隆到杆状病毒转移载体pFastBac-dual(pFBd)中,获得相应的重组转移质粒;转化大肠杆菌菌株DH10bac,转座后经筛选获得重组穿梭质粒rbacmid,将重组穿梭质粒转染孔板培养的Sf9细胞,获得含人RBP4表达框的重组杆状病毒,经过扩增获得毒种。毒种感染对数生长期的Sf9细胞并表达人RBP4蛋白(I-RBP4),通过SDS-PAGE和Western blotting对表达蛋白进行检测和鉴定。用毒种感染悬浮培养的Sf9细胞制备一批RBP4蛋白,完成SDS、Western blotting的检测及少量的多抗制备。纯化重组蛋白并与E.coli重组人RBP4(E-RBP4)分别免疫家兔。实验结果,酶切鉴定及测序证实重组转移质粒构建正确;成功构建重组RBP4-bacmid;人RBP4蛋白在昆虫细胞获得高效表达。表达的RBP4蛋白可以分泌到培养基中,分子量约为23 kDa,经过计算表达量为100 mg/L;纯化蛋白免疫兔子制备了多抗血清,血清滴度为1∶100 000,高于原核表达的抗体滴度(1∶10 000),与人体提纯蛋白制备的抗体滴度相近。杆状病毒系统高效表达了人的RBP4蛋白,具有较好的抗原性,并获得高亲和力的抗血清,为下一步的人血RBP4检测试剂盒的制备打下了坚实的基础。     

Expression and purification of the angiogenesis inhibitor 16-kDa prolactin fragment from insect cells

The 16-kDa fragment of prolactin (16-kDa PRL), derived from proteolytic cleavage of 23-kDa PRL, was shown to have antiangiogenic activity. Previous studies have shown that recombinant 16-kDa PRL produced from bacteria often contained endotoxins, which are cytotoxic to endothelial cells, and varied in its biological activity due to changes in its refolding from inclusion bodies. These problems limited the use of recombinant 16-kDa PRL. To improve the generation of recombinant 16-kDa PRL, we expressed 16-kDa PRL in Sf9 insect cells using a baculoviral expression system. The signal sequence of the human PRL gene and codons for seven histidines were added to the N- and C-termini, respectively, of the 16-kDa PRL cDNA construct. Recombinant 16-kDa PRL was detected in both the cell pellet and the medium. About 0.28 mg purified protein was isolated from the cell pellet of 4 x 10(7) infected cells using nickel affinity chromatography. Sixteen kilodalton PRL was posttranslationally modified with apparent molecular weights of 16 and 18 kDa on SDS-PAGE. The level of 18-kDa protein was significantly reduced after digestion with peptidyl-N-glycosidase, suggesting that the heterogeneity was due to glycosylation of 16-kDa PRL. N-terminal sequence analysis confirmed the fact that both proteins were human 16-kDa PRL and the signal sequences were cleaved at the same position as that of human PRL. Consistent with its role as an angiogenesis inhibitor, purified recombinant 16-kDa PRL inhibits the proliferation of endothelial cells with a potency similar to that previously reported for the protein generated in Escherichia coli. This 16-kDa PRL expressed in Sf9 cells is a useful reagent for functional studies and for the purification and identification of its receptor.     

Overexpression and characterization of the human mineralocorticoid receptor.

The full-length human renal mineralocorticoid receptor (hMR) has been overproduced in Spodoptera frugiperda (Sf9) insect cells using baculovirus-mediated expression. The overproduced hMR binds aldosterone with high affinity (Kd = 1.36 nM) and has high affinity for cortisol, cortexolone, and progesterone. Immunoprecipitation and immunoblot analysis of the recombinant hMR with MR-specific antibodies reveal three major protein bands with molecular masses of 115, 119, and 125 kDa. hMR isoforms show maximal accumulation at 48 h post-infection with the recombinant baculovirus. Maximal aldosterone binding was detected at 24 h rather than at 48 h post-infection, suggesting that the assembly of hMR monomers into the nonactivated steroid-binding receptor complexes and/or their stability deteriorates after 24 h post-infection. It is estimated by specific aldosterone binding that 1.2 x 10(6) hMR molecules are expressed per Sf9 cell (equivalent to 7 pmol/mg of cytosolic protein) at 24 h post-infection. 5-Fold more receptor molecules/cell are expressed but not detected by steroid binding at 48 h post-infection as determined by immunoblot analysis. Using the MR-specific H10E anti-idiotypic monoclonal antibody, immunoprecipitation of cytosol from recombinant baculovirus-infected Sf9 cells pulse-labeled with 32Pi demonstrated for the first time that the recombinant hMR is highly phosphorylated. The hMR is expressed as 9-10 S oligomeric complexes (Stokes radii approximately 67-85 A) that are slightly heavier than the unactivated glucocorticoid receptor and can be converted to smaller 4 S receptor monomers (Stokes radii approximately 25-55 A) by elevated temperature, pH, and ionic strength. Unlike the glucocorticoid receptor, the oligomeric hMR complex can bind DNA-cellulose without prior activation. Finally, indirect immunofluorescence demonstrated that the hMR is expressed primarily as a cytoplasmic protein that can be induced to translocate to the nucleus upon treatment with hormone.     

High-Level Synthesis and Fate of Acetylcholine in Baculovirus-Infected Cells: Characterization and Purification of Recombinant Rat Choline Acetyltransferase

Rat choline acetyltransferase (ChAT) has been expressed at a high level in Spodoptera frugiperda Sf9 cells using a baculovirus expression system. A cDNA containing the coding sequence for ChAT was inserted into the transfer vector pAcYM1 to yield the recombinant vector pAcYM1/ChAT. Sf9 cells were then coinfected with pAcYM1/ChAT and the wild-type Autographa californica virus. One recombinant virus particle, containing the cDNA for ChAT, was selected that expressed a protein of 68.5 kDa. Forty hours after infection of cells with the recombinant virus, the specific activity of ChAT in the cytosol was 190 nmol of acetylcholine/min/mg of protein, accounting for approximately 24% of the cell cytosolic proteins as being ChAT. The apparent Km values of the enzyme for choline and acetyl-CoA were 299 and 221 microM, respectively, whereas the respective Vmax values were 10.6 and 11.4 mumol of acetylcholine/min/mg of protein. In addition, analysis of the protein revealed that ChAT is phosphorylated in Sf9 cells. About 0.5 mg of ChAT was obtained from a one-step purification procedure starting with 10(8) infected Sf9 cells. Addition of choline to the incubation medium led to accumulation of high amounts of acetylcholine in the cytosol of the infected cells. The neurotransmitter was not released by Sf9 cells in response to membrane depolarization or on ionophore-mediated calcium entry. Some acetylcholine, which most likely originated from cell death inherent to viral infection, accumulated in the culture medium. The infected insect cells, which synthesize and store neurotransmitter, provide a new and convenient model for analyzing synaptic transmission at the molecular level.     

A GP64-null baculovirus pseudotyped with vesicular stomatitis virus G protein

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein is an essential virion protein that is involved in both receptor binding and membrane fusion during viral entry. Genetic studies have shown that GP64-null viruses are unable to move from cell to cell and this results from a defect in the assembly and production of budded virions (BV). To further examine requirements for virion budding, we asked whether a GP64-null baculovirus, vAc(64-), could be pseudotyped by introducing a heterologous viral envelope protein (vesicular stomatitis virus G protein [VSV-G]) into its membrane and whether the resulting virus was infectious. To address this question, we generated a stably transfected insect Sf9 cell line (Sf9(VSV-G)) that inducibly expresses the VSV-G protein upon infection with AcMNPV Sf9(VSV-G) and Sf9 cells were infected with vAc(64-), and cells were monitored for infection and for movement of infection from cell to cell. vAc(64-) formed plaques on Sf9(VSV-G) cells but not on Sf9 cells, and plaques formed on Sf9(VSV-G) cells were observed only after prolonged intervals. Passage and amplification of vAc(64-) on Sf9(VSV-G) cells resulted in pseudotyped virus particles that contained the VSV-G protein. Cell-to-cell propagation of vAc(64-) in the G-expressing cells was delayed in comparison to wild-type (wt) AcMNPV, and growth curves showed that pseudotyped vAc(64-) was generated at titers of approximately 10(6) to 10(7) infectious units (IU)/ml, compared with titers of approximately 10(8) IU/ml for wt AcMNPV. Propagation and amplification of pseudotyped vAc(64-) virions in Sf9(VSV-G) cells suggests that the VSV-G protein may either possess the signals necessary for baculovirus BV assembly and budding at the cell surface or may otherwise facilitate production of infectious baculovirus virions. The functional complementation of GP64-null viruses by VSV-G protein was further demonstrated by identification of a vAc(64-)-derived virus that had acquired the G gene through recombination with Sf9(VSV-G) cellular DNA. GP64-null viruses expressing the VSV-G gene were capable of productive infection, replication, and propagation in Sf9 cells.     

Pancreatic cholesterol esterases. 1. Pancreatic cholesterol esterase induction during maturation

The activities of pancreatic cholesterol esterase from calf and cow pancreas were examined in detail. A 1300-fold enhancement of enzymatic activity was found after maturation, even though cholesterol esterase activity levels in other organs did not change from the juvenile to the adult species. Radioimmunoassays also showed that the calf pancreas contained at least 100-fold less cholesterol esterase protein. Decreased amounts of protein were not due to enhanced proteolysis, since cytosol from cow pancreas degrades exogenously added cholesterol esterase faster than that from calf pancreas. Rather, enhancement of pancreatic cholesterol esterase activity associated with bovine maturation was the result of specific, increased synthesis of a 72-kDa enzyme. This labile 72-kDa cholesterol esterase species was purified to homogeneity by a two-step process in 75% yield and is the major form of bovine pancreatic cholesterol esterase (99%). A much less abundant 67-kDa species, accounting for less than 1% of total pancreatic cholesterol esterase activity, was also purified to homogeneity in a similar two-step process. These results demonstrate that a specific form of pancreatic cholesterol esterase is induced during maturation, and they bear importantly on understanding juvenile cholesterol metabolism as related to dietary absorption of this sterol.     

Expression in Spodoptera frugiperda (Sf21) insect cells of BT-R(1), a cadherin-related receptor from Manduca sexta for Bacillus thuringiensis Cry1Ab toxin

The cadherin-related receptor of Manduca sexta, BT-R(1), for the Cry1A family of Bacillus thuringiensis insecticidal toxins, was expressed in cultured Spodoptera frugiperda (Sf21) insect cells utilizing the expression vector deltaOp-gp64. Recombinant BT-R(1) was released by the Sf21 cells in soluble form into the culture medium and represents approximately 58% of total BT-R(1) produced by the cells. The soluble protein was purified by affinity chromatography using Cry1Ab toxin coupled to Sepharose 4B. The apparent molecular mass of purified soluble recombinant BT-R(1) is 195 kDa. Radiolabeled toxin bound to purified soluble BT-R(1) with a K(d) value of 1.1 nM, which is similar to that of both membrane-bound BT-R(1) in Sf21 cells and natural BT-R(1) from M. sexta larval midgut tissue. Binding of radiolabeled toxin to soluble BT-R(1) was competitively inhibited by unlabeled Cry1Ab toxin but not by other Cry toxins as was observed also for membrane-bound BT-R(1). The recombinant soluble protein was stable in culture medium for at least 3 days at 27 degrees C and for 7 days at 4 degrees C and exhibited toxin-binding properties similar to the natural protein. Apparently, neither membrane association nor the extent of glycosylation influences the binding affinity and specificity of BT-R(1). Approximately 1 mg of purified BT-R(1) was obtained per liter of insect cell culture supernatant, representing approximately 2 x 10(9) Sf21 cells.     

Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system.     

Chemical and biological activities of a 64-kilodalton outer sheath protein from Treponema denticola strains.

This study examined the distribution of the major outer sheath proteins (MOSP) in several Treponema denticola strains and reports the isolation of a 64-kDa protein from the outer sheath of human clinical isolate T. denticola GM-1. The outer sheath was isolated by freeze-thaw procedures, and the distribution of outer sheath proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). T. denticola GM-1, MS25, SR-5, and three low-passage clinical isolates possessed an MOSP with a relative molecular mass of 60 to 64 kDa. This MOSP was absent in T. denticola ATCC 35404 (TD-4) and clinical isolate SR-4. The latter possessed an MOSP of 58 kDa. 125I labeling revealed both MOSP to be dissociated forms of higher-molecular-mass oligomeric units between 116 and 162 kDa. Two-dimensional SDS-PAGE confirmed the modifiability of these MOSP. Isoelectric focusing of the 64-kDa MOSP indicated a pI of 6.7. Immunoblots with antiserum to GM-1 whole cells revealed the 64-kDa protein to be immunogenic and not cross-reactive with the MOSP of TD-4 or SR-4, and monospecific antibody to the 64-kDa protein recognized common epitopes on the high-molecular-weight oligomeric protein. These antibodies did not react with any component of TD-4 whole cells in immunoblots or in immunogold electron microscopy. Fab fragments inhibited the adherence of T. denticola GM-1 to human gingival fibroblasts by 78% (1:1,600; 0.72 micrograms of protein per ml), while TD-4 adherence was not inhibited. Amino acid analysis revealed a slightly acidic protein, devoid of cysteine, with 36% hydrophobic residues. Cyanogen bromide fragmentation of the 64-kDa protein revealed that a 42-kDa fragment contained a T-L-D-L-A-L-D segment which was 100% homologous with an integrin alpha subunit of a human leukocyte adhesion glycoprotein p 150,95.     

Uptake of chylomicron remnants causes cholesterol accumulation in cultured human arterial smooth muscle cells

Chylomicron remnants (Sf greater than 100) were prepared by treating human chylomicrons (Sf greater than 400) with human post heparin plasma. Chylomicron remnants recovered after 70-80% of chylomicron triacylglycerol was hydrolyzed, suppressed LDL-receptor activity and increased cell cholesterol esterification to the same extent as did LDL when added to cultured human arterial smooth muscle cells at an equal cholesterol concentration. Cell cholesterol mass increased 36% after incubation with 25 micrograms LDL cholesterol/ml and 35% with 25 micrograms chylomicron-remnant cholesterol/ml. Addition of 30 microM chloroquine plus LDL or chylomicron remnants further increased cholesterol content of cells (74% and 87%, respectively) and caused a significant rise in cell esterified cholesterol (344% and 369%, respectively). Cholesterol content per unit of apolipoprotein B mass of remnants was 2-3-fold higher than that of LDL. Therefore, if lipoprotein particles were added at equivalent apolipoprotein B mass chylomicron remnants increased cell cholesterol content and cholesterol esterification and suppressed LDL receptor activity significantly more than did LDL. This suggests that an additional determinant, presumably apolipoprotein E, is important for receptor recognition of chylomicron remnants. These results may be relevant to the delivery of chylomicron-derived cholesterol to arterial cells proposed as a feature of atherogenesis.     

人骨保护素-热休克蛋白70融合蛋白在昆虫细胞中的表达和活性分析

目的:利用BaculoDirect杆状病毒表达系统融合表达人OPG功能片段p22-194和分枝杆菌HSP70 p111-125基因,并鉴定重组蛋白及其生物学活性。方法:将编码人OPG功能片段和分枝杆菌HSP70功能片段基因克隆至杆状病毒转座载体,将重组转座载体与BaculoDirectTM Linear DNA进行LR重组连接反应,构建出重组杆状病毒DNA,转染Sf9昆虫细胞,获得重组病毒。在Sf9细胞中进行表达,并对表达产物进行SDS-PAGE电泳、Western blotting分析,用Ni柱纯化。采用破骨细胞生成抑制试验和抑炎试验鉴定表达产物的生物学活性。结果:重组病毒在感染昆虫细胞后48h开始出现一相对分子质量为28 kDa大小的特异条带,感染后72～96 h蛋白量达到高峰。破骨细胞生成抑制实验及抑炎试验结果显示,重组蛋白能明显抑制破骨细胞的生长和分化,同时亦具有抑制炎症反应的作用。结论:利用杆状病毒表达系统在昆虫细胞中成功表达OPG-HSP70融合蛋白,该融合蛋白具有抑制破骨细胞生成和抑制炎症反应生物学活性。     

Study on persistent infection of Japanese encephalitis virus Beijing-1 strain in serum-free Sf9 cell cultures

Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8 x 10(6) cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0 x 10(5) to 1.5 x 10(6) pfu/ml. The infected Sf9 cells could be sub-cultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.     

Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic

Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40 kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.     

Isolation and characterization of a Mr = 38,000 protein from differentiating smooth muscle cells

In culture, vascular smooth muscle cells grow and form a confluent monolayer of cells. Under appropriate conditions, regions of the monolayer can be induced to draw away from the substrate and form multicellular nodules. The ultrastructure of the cells in the nodules appears to be similar to that of differentiated smooth muscle cells. The process of nodulation is associated with the synthesis of a unique protein whose molecular weight is estimated from gradient gel electrophoresis to be 38,000 (38-kDa Protein). The protein is secreted into the culture medium and can be detected either by metabolic labeling or by staining with Coomassie Blue. Partial purification of 38-kDa Protein was achieved using affinity chromatography. The protein is adsorbed to heparin-agarose, but not to gelatin-agarose. The concentration of 38-kDa Protein in nodular conditioned medium is estimated at 1.9 micrograms/ml and less than 0.01 microgram/ml in conditioned medium made from monolayer cells. The presence of 5% fetal bovine serum in the labeling medium does not affect 38-kDa Protein synthesis. Cross-reactivity with fibronectin was evaluated using polyvalent antibodies to 38-kDa Protein. The 38-kDa Protein is not antigenically related to fibronectin. Furthermore, we establish that the protein is not qualitatively influenced by the presence of ascorbate (50 micrograms/ml), beta-aminoproprionitrile fumarate (50 micrograms/ml) heparin (10 ng/ml), or fibronectin (20 micrograms/ml) in the culture medium. We find that the added components neither suppress 38-kDa Protein synthesis in nodular cultures nor enhance 38-kDa Protein synthesis in monolayer cultures. The 38-kDa Protein is not detected in either monolayer or nodular cell layers and appears to be a secreted protein. Its appearance in nodular conditioned medium during nodulation suggests a relationship with that process.     

Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm

Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.