Production of proteoglycans by human lung fibroblasts (IMR-90) maintained in a low concentration of serum.

Maintenance of fibroblasts in 0.5% serum results in viable but non-proliferative cells that may be analogous to fibroblasts in vivo. The synthesis of proteoglycans by human embryo lung fibroblasts in Eagle's minimal essential medium with 0.5% newborn-bovine serum or with 10% serum has been compared. A similar amount of [35S]sulphate-labelled glycosaminoglycan per cell was secreted by fibroblasts in 10% or 0.5% serum. 35SO42-incorporation into sulphated glycosaminoglycans was enhanced in 0.5% serum when expressed per mg of cell protein, but [3H]glucosamine incorporation was decreased. The charge density of these glycosaminoglycans was not changed as determined by ion-exchange chromatography. It was concluded that decreased protein/ cell resulted in an apparent increase in 35S-labelled glycosaminoglycan synthesis/mg of cell protein, whereas decreased uptake of [3H]glucosamine resulted in a decrease in their glucosamine labelling. The proteoglycans secreted by fibroblasts in 0.5% serum were similar in glycosaminoglycan composition, chain length and buoyant density to the dermatan sulphate proteoglycan, which is the major secreted component of cells in 10% serum. Larger heparan sulphate and chondroitin sulphate proteoglycans, which comprise about 40% of the total secreted proteoglycans of cultures in 10% serum, were greatly diminished in the medium of cultures in 0.5% serum. The proteoglycan profile of medium from density-inhibited cultures in 10% serum resembles that of proliferating cultures, indicating that lack of proliferation was not responsible for the alteration. The dermatan sulphate proteoglycan, participating in extracellular matrix structure, may be the primary tissue product of lung fibroblasts in vivo.     

Characterization of lysozyme synthesized by differentiated mouse myeloid leukemia cells.

Lysozyme was induced by dexamethasone during normal differentiation of cultured mouse myeloid leukemia cells (M1) to macrophages and granulocytes. A large amount of lysozyme was produced by macrophage-like line cells (Mm-1), established from spontaneously differentiated macrophage-like cells from a clonal line of M1 cells. Lysozyme purified from the culture medium of these Mm-1 cells (Mm-1 lysozyme) had a molecular weight of 15,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and showed maximal activity at pH 6.6 with an optimal NaCl concentration of 0.04 M. Its mobility on polyacrylamide gel electrophoresis at pH 4.5 was distinctly lower than those of lysozymes from hen egg white and human urine. Rabbit anti-Mm-1 lysozyme serum inhibited the activities of lysozyme preparations from peritoneal macrophages of normal mice and rats and dexamethasone-induced differentiated M1 cells, but not those of preparations from hen egg white and human urine. Lysozyme was also purified from normal mouse lung, which is rich in alveolar macrophages and was found to be similar to lysozyme purified from the culture medium of Mm-1 cells in size and electrophoretic mobility and in its pH optimum, trypsin peptide map, and antigenicity. Thus the molecular structure of the lysozyme induced in differentiated mouse myeloid leukemia cells is similar to that of lysozyme produced by normal cells.     

Demonstration of pancreatic secretory trypsin inhibitor in serum-free culture medium conditioned by the human pancreatic carcinoma cell line CAPAN-1

Serum-free culture medium conditioned by an established human pancreatic adenocarcinoma cell line, CAPAN-1, contains copious amounts of immunoreactivity due to pancreatic secretory trypsin inhibitor (PSTI) as demonstrated by radioimmunoassay. The immunoreactive substance was purified from the conditioned medium to apparent homogeneity by trypsin affinity and gel filtration chromatography with an overall recovery of 40%, and its primary structure was determined by Edman degradation. The immunoreactive substance is a peptide of 56 amino acid residues with a calculated molecular weight of 6,241. Its amino acid composition, primary structure, and inhibitory effect against trypsin are indistinguishable from those of human pancreatic juice PSTI, indicating that this substance is PSTI itself. This is the first direct demonstration that tumor cells secrete PSTI in vitro. When CAPAN-1 was inoculated into a nude mouse, it produced a tumor and the tumor synthesized human PSTI in vivo, as demonstrated by the fact that the tumor extract contained 99.0 +/- 26.2 ng of human PSTI/mg of protein, while PSTI was not detected in extracts from other tissues examined. Furthermore, high levels of human PSTI (14.3 +/- 2.6 ng/ml) were detected in the serum of tumor-bearing mice but not in that of nontumor-bearing mice, suggesting that PSTI secreted from the tumor appears in the blood circulation. Taken together, these results strongly support the view that the serum levels of PSTI are elevated in cancer-bearing patients due to secretion of this peptide from tumor cells per se.     

Expression, purification, and characterization of humanized anti-HBs Fab fragment

Anti-HBs Fab fragment has considerable potential for use in the prevention and treatment of liver diseases by HBV. Here we established a high-level expression system to directly produce anti-HBs Fab fragment in Pichia pastoris. This was achieved by co-integration of the genes encoding the heavy and light chains both under the genome of the yeast cells. The Fab fragment was efficiently secreted into medium at a concentration of 50 mg/liter. The authenticity of the Fab fragment was confirmed by immunoblot analysis, which yielded one band of approximately 50 kDa under nonreducing conditions and two bands of approximately 28 kDa under reducing conditions. The anti-HBs Fab fragment was prepared with a purity of 95% by affinity chromatography. The affinity activity of the recombinant Fab was detected by ELISA, which indicated that 1 mg of recombinant Fab was equivalent to 40 IU HBIG (20 IU/mg). The results demonstrated that the recombinant Fab fragment could sufficiently neutralize the HBsAg.     

Active Recombinant Rat Dipeptidyl Aminopeptidase I (Cathepsin C) Produced Using the Baculovirus Expression System

An active form of rat dipeptidyl aminopeptidase I (DPPI, cathepsin C) was obtained by heterologous expression in insect cells. Baculoviruses carrying a cDNA sequence encoding the entire rat DPPI precursor was used to infect High Five cells in a serum-free medium. Recombinant DPPI (rDPPI) was secreted into the medium from which it was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography (HIC), and ion-exchange chromatography. A polyhistidine-tagged form of the enzyme (HT-rDPPI) was purified from the medium by immobilized metal affinity chromatography (IMAC).In vivoactivation of native rat DPPI involves at least three chain cleavages per subunit and the ability of the expression system to imitate this processing was investigated. Both rDPPI and HT-rDPPI were secreted into the medium as unprocessed and inactive proenzymes and gradually converted into their active forms in the medium. This process was not completed at the time of harvest but mature enzyme processed similarly to native rat and human DPPI could be obtained by incubating the eluates from the HIC and IMAC columns at pH 4.5 and 5°C for 18–40 h. The yield of purified and matured enzyme was approximately 50 mg/liter, and it was shown that rDPPI and HT-rDPPI were active against both a dipeptide–p-nitroanilide substrate and human growth hormone N-terminally extended with an Ala-Glu dipeptide.     

Purification and characterization of a plasminogen activator secreted by a pig kidney cell line

The plasminogen activator secreted by calcitonin-treated pig kidney cells was purified, characterized and compared with human urinary urokinase. The purification procedure was based on the following steps: sulphopropyl-Sephadex chromatography, p-aminobenzamidine-Sepharose chromatography, preparative sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectrofocusing. The purified enzyme was obtained from the conditioned medium with a yield of 13% and a purification factor of 390-fold. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under non-reducing conditions showed one closely spaced doublet with an Mr of 50 000; in the presence of reducing agents, two additional bands of Mr 30 000 and 20 000 appeared. The purified enzyme resembles the 53 000-Mr components of human urinary urokinase in amino acid composition and two-dimensional tryptic peptide maps and in its catalytic properties, and the two enzymes cross-react immunologically with rabbit antibodies raised against either. The enzyme appears to be different from tissue plasminogen activator secreted by HeLa cells.     

Yeast mutants with enhanced ability to secrete human lysozyme: Isolation and identification of a protease-deficient mutant

Summary Yeast mutant strains which secrete large amounts of human lysozyme were screened using an agar medium containing bacterial cells. Nine mutants secreted over 10 times more lysozyme than the wild-type parent strain. The mRNA levels for lysozyme in the mutants were not higher than that of the wild-type strain. Three of the mutant strains were deficient in carboxypeptidase Y activity. It was found that the protease deficiency was caused by a deficiency in conversion of proenzyme to mature enzyme in ssl1 mutant cells. The ssl1 gene was found to be closely linked to the centromere and determine both the efficiency of secretion of lysozyme and the processing of carboxypeptidase Y.Abbreviations CPY carboxypeptidase Y (yscY) - HLY a synthetic gene for human lysozyme     

人工合成表皮生长因子基因在甲基营养型酵母中的高效表达

选用酵母菌偏爱密码子人工合成了编码５１个氨基酸的人表皮生长因子（ｈＥＧＦ）基因．将合成基因与编码酵母α因子前导肽８５个氨基酸的ＤＮＡ片段融合后克隆到醇氧化酶基因启动子下游,并构建出多拷贝表达载体．此载体转化甲基营养型酵母株ＧＳ１１５后筛选出整合型ＭｕｔＳＨｉｓ＋基因型菌株．高密度培养及诱导表达后该株可分泌具完好生物活性和正确物理化学性质的人表皮生长因子,产量达１００ｍｇ／Ｌ,经３次柱层析纯度达９５％以上,为观察其生物学作用打下了良好基础     

Heterologous protein secretion by Aspergillus niger growing in submerged culture as dispersed or aggregated mycelia

The secreted production of a heterologous enzyme, hen egg-white lysozyme, by Aspergillus niger was studied in shake flasks containing media of different initial viscosities. Raising the viscosity of the medium by addition of polyvinylpyrrolidone (PVP) brought about a transition in the form of growth from aggregated mycelia (pellets) to dispersed mycelia. The specific yield of lysozyme in cultures containing an initial concentration of 5% (w/v) starch was 8 mg lysozyme/g dry weight. Addition of 2% (w/v) PVP to the medium resulted in a specific yield of 14 mg lysozyme/g dry weight.     

Expression, purification, and characterization of the recombinant calcium-binding equine lysozyme secreted by the filamentous fungus Aspergillus niger: comparisons with the production of hen and human lysozymes.

Equine lysozyme (EqL) has been expressed from a synthetic gene and secreted from a heterologous host, the filamentous fungus Aspergillus niger. By including 100 mM Ca2+ in the growth medium, secreted yields of more than 50 mg/liter could be achieved using polyvinylpyrrolidone (PVP) complete medium. In a soya medium yields of up to 150 mg/liter were achieved. The production of recombinant human lysozyme (HuL) from A. niger with yields of over 40 mg/liter was also achieved using PVP medium. Addition of Ca2+ to the growth medium reduced the yield of both HuL and hen egg white lysozyme (HEWL). Sequence differences between the three lysozymes, EqL, HuL, and HEWL, resulted in different susceptibilities to cleavage by A. niger proteases. An improved procedure for the purification of EqL and HuL from A. niger allowed separation of the proteins from pigments produced by the fungus. Detailed spectroscopic analysis, including 2D 1H NMR, for recombinant EqL and recombinant HuL confirm that both proteins possess their native structure and are purified to homogeneity.     

Expression,purification, and characterization of an unstable lysozyme mutant in Pichia pastoris

To investigate the expression and purification of an unstable heterologous protein in Pichia pastoris, the cDNA of H5-lysozyme, a hen egg lysozyme mutant with a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) fused to the carboxyl terminus, was integrated into the genome of P. pastoris. It was found that medium composition, induction time, and fermenter type were important factors for the expression of H5-lysozyme. Substantially active H5-lysozyme was secreted by induction with methanol when the prepro-sequence of alpha-factor was used as secretion signal sequence. The amount secreted was 422-fold greater than that observed with Saccharomyces cerevisiae. Recombinant H5-lysozyme was recovered and purified by cation-exchange chromatography directly from fermentation broth. The mutant lysozyme showed bactericidal activity against Gram-positive as well as Gram-negative bacteria.     

Differences between Saccharomyces cerevisiae and Bacillus subtilis in secretion of human lysozyme

Saccharomyces cerevisiae secreted human lysozyme in the medium as an active form when the signal peptides of chicken lysozyme and a chicken lysozyme-Aspergillus awamori glucoamylase hybrid were used, whereas it did not synthesize any human lysozyme protein by using the signal peptide of A. awamori glucoamylase. The secreted lysozyme was easily purified and crystallized. On the other hand, Bacillus subtilis secreted an inactive human lysozyme, which seemed to have incorrect disulfide bonds, with the signal peptide of amylase and its mutants. The free energy changes for the membrane translocation of the signal peptides are related to the secretion of human lysozyme in S. cerevisiae, but not in B. subtilis. These results indicate that differences exist between S. cerevisiae and B. subtilis in the secretion of human lysozyme.     

High-level, heat-regulated synthesis of proteins in eukaryotic cells

Plasmids have been constructed in which promoters of 70-kDa heat-shock protein genes (hsp70) of human and Drosophila origin were linked to three different eukaryotic genes encoding human growth hormone (hGH), chicken lysozyme (cL) and a human influenza haemagglutinin (HA). Following transfection into widely divergent eukaryotic cells, the hybrid genes direct the transient, heat-regulated synthesis of the three proteins. hGH and cL are secreted into the medium. A human hsp70-hGH construct was used to establish stable mouse fibroblast lines that are capable of producing and secreting hGH at high levels following heat induction: hGH is secreted at a 500-1200-fold higher rate by heat-treated than by untreated cells.     

Human lysozyme: sequencing of a cDNA, and expression and secretion by Saccharomyces cerevisiae

A cDNA encoding human lysozyme was isolated from a human placenta cDNA library. The cDNA was 1.5 kb in size and coded for a signal peptide consisting of 18 amino acids and mature lysozyme. The amino acid sequence of the mature lysozyme, deduced from the nucleotide sequence, was identical with the published sequence. In the 3'-noncoding region of the cDNA, an Alu sequence was found in the reverse orientation. In a protein coding region, the human lysozyme cDNA shows 60.1% and 51.3% similarity with chicken lysozyme and human alpha-lactalbumin cDNAs, respectively. When the cDNA was expressed in Saccharomyces cerevisiae, an active and correctly processed human lysozyme was secreted efficiently into the culture medium.     

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.     

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.     

Characterisation of two differently processed forms of human recombinant factor IX synthesised in CHO cells transformed with a polycistronic vector

A stable transformed cell line constitutively expressing human factor IX has been established. Wild-type Chinese hamster ovary cells (CHO cells) were transformed using a polycistronic expression vector carrying a previously isolated factor IX cDNA and a selection gene encoding the Escherichia coli xanthine-guanine phosphoribosyl transferase. One clone, CHO 622.4, contains a high number of genomically integrated plasmids and secretes 1-3 mg factor IX l-1 day-1 into the culture medium with a biological activity ranging from 25% to 40%. The recombinant molecule was purified either by conventional chromatography or by immunoaffinity chromatography using antibodies specific to a calcium-induced factor IX conformer. The purified recombinant protein migrates as a single band with the same mobility as that of natural factor IX on SDS/polyacrylamide gels. N-terminal sequencing shows tow differently processed forms of recombinant factor IX: whereas the majority of the zymogen is correctly processed, approximately 20% of the purified recombinant molecule contains an 18-amino-acid NH2-extension corresponding to the precursor form of factor IX. Analysis of the 4-carboxyglutamic acid content indicates a high but incomplete carboxylation (70%) of the recombinant molecule as compared to natural factor IX. The carbohydrate composition of both the natural and recombinant molecules has been determined. Both molecules have a N-glycan structure of similar complexity, indicating that factor IX contains all the information to direct the same glycosylation pattern in human liver cells and in an unrelated cell line such as CHO-K1.     

Coisolation of glutathione peroxidase, catalase and superoxide dismutase from human erythrocytes

Glutathione peroxidase (GSH-Px; glutathione: hydrogen peroxide oxidoreductase; EC 1.11.1.9), catalase (H2O2: H2O2 oxidoreductase; EC 1.11.1.6) and superoxide dismutase (superoxide: superoxide oxidoreductase; EC 1.15.1.1) were coisolated from human erythrocyte lysate by chromatography on DEAE-cellulose. Glutathione peroxidase was separated from superoxide dismutase and catalase by thiol-disulfide exchange chromatography and then purified to approximately 90% homogeneity by gel permeation chromatography and dye-ligand affinity chromatography. Catalase and superoxide dismutase were separated from each other and purified further by gel permeation chromatography. Catalase was then purified to approximately 90% homogeneity by ammonium sulfate precipitation and superoxide dismutase was purified to apparent homogeneity by hydrophobic interaction chromatography. The results for glutathione peroxidase represent an improvement of approximately 10-fold in yield and 3-fold in specific activity compared with the established method for the purification of this enzyme. The yields for superoxide dismutase and catalase were high (45 mg and 232 mg, respectively, from 820 ml of washed packed cells), and the specific activities of both enzymes were comparable to values found in the literature.     

Lipid composition of PC12 pheochromocytoma cells: characterization of globoside as a major neutral glycolipid

We have studied the lipid composition of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF). Neutral and acidic lipid fractions were isolated by column chromatography on DEAE-Sephadex and analyzed by high-performance thin-layer chromatography (HPTLC). The total lipid concentration was approximately 220 micrograms/mg of protein, and the concentration of neutral glycolipids was 1.6-1.8 microgram/mg of protein for both NGF-treated and untreated cells. The neutral glycolipid fraction contained a major component, which accounted for approximately 80% of the total and which was characterized as globoside on the basis of HPTLC mobility, carbohydrate analysis, fast atom bombardment mass spectrometry, and mild acid hydrolysis. The major fatty acids of globoside were C16:0 (10%), C18:0 (16%), C22:0 (23%), C24:1 (17%), and C24:0 (24%). C18 sphingenine accounted for almost all of the long-chain bases. The other neutral glycolipids were tentatively identified as glucosylceramide (15%), lactosylceramide (4%), and globotriosylceramide (4.5%). The concentration of ganglioside sialic acid was approximately 0.34 and 0.18 microgram/mg of protein for cells grown in the presence and absence of NGF, respectively. Although there was an increase in ganglioside concentration in NGF-treated cells, NGF did not produce any differential effects on the relative proportions of the individual gangliosides. Several of the gangliosides appear to contain fucose, and one of these was tentatively identified as fucosyl-GM1. Brain-type gangliosides of the ganglio series were also detected by an HPTLC-immunostaining method. However, the fatty acid and long chain base compositions of PC12 cell gangliosides (and their TLC mobility) differ from those of brain gangliosides.(ABSTRACT TRUNCATED AT 250 WORDS)