Immunoaffinity purification of human prorenin produced in Chinese hamster ovary cells

A simple immunoaffinity column chromatographic procedure is described whereby recombinant human prorenin secreted from Chinese hamster ovary cells may be isolated in a high state of purity from serum-free culture medium. Prorenin thus purified has been characterized by SDS-polyacrylamide gel electrophoresis and by partial sequence analysis which has revealed the expected N-terminal sequence. Trypsin treatment gives rise to renin, and reversible acid activation has also been demonstrated for the recombinant zymogen.     

Isolation and characterization of recombinant human prorenin in Chinese hamster ovary cells

Recombinant human prorenin (rh-prorenin) was purified from supernatants of Chinese hamster ovary (CHO) cell line transfected with the cDNA for rh-prorenin by employing a simple two-step procedure which consisted of ammonium sulfate precipitation and immunoaffinity chromatography using a monoclonal antibody specific for the profragment of human prorenin. About 100-fold purification with 35% recovery was achieved after the two steps. Purified rh-prorenin migrated as a single protein band with apparent molecular weights of 46,000-47,000 and about 50,000 on SDS-PAGE and gel filtration (HPLC), respectively, although it consisted of multiple components (pI values, 5.6-6.4) that could be resolved by isoelectric focusing (IEF). The treatment of rh-prorenin with endo-beta-N-acetylglucosaminidase converted the rather broad protein band to a sharp band on SDS-PAGE and reduced the number of multiple pI peaks on IEF. Amino-terminal sequence analysis of both the purified rh-prorenin and rh-renin revealed Leu-Pro-Thr-Asp- and Leu-Thr-Leu-Gly-, respectively, which agreed with those predicted from the base sequences of their cDNA. These data suggested that microheterogeneity of rh-prorenin is due to the carbohydrate moiety, but not to the protein moiety. Purified rh-prorenin was almost inactive, but was cleaved at the carboxyl end of a dibasic pair Lys-2-Arg-1 by trypsin and converted to active renin. However, at the early stage during trypsin activation, new intermediate forms between rh-prorenin and rh-renin were formed, suggesting multiple activation steps of rh-prorenin in addition to the one step activation.     

Characterization of the oligosaccharide structures on recombinant human prorenin expressed in Chinese hamster ovary cells.

Prorenin was isolated by immunoprecipitation from the culture medium of Chinese hamster ovary cells transfected with a human prorenin cDNA. The N-linked oligosaccharide structures on the in vivo [3H]mannose-labeled, purified protein were characterized using a combination of serial lectin affinity chromatography, high-pressure liquid chromatography, ion-exchange chromatography, and size-exclusion chromatography and treatment with specific glycosidases and methylation analysis. Approximately 61% of the oligosaccharides on the molecule are complex type, in the form of tetraantennary (2%), 2,6-branched triantennary (13%), 2,4-branched triantennary (3%), and biantennary (43%) structures. The majority of all complex type structures are core-fucosylated. Sialic acids are linked at the C-3 position of terminal galactose, and the degree of sialylation of the bi- and triantennary structures varies between nonsialylated and fully sialylated; no tetraatennary structure contains more than three sialic acid residues. Recombinant prorenin contains 4% hybrid-type structures, all of which carry a terminal sialic acid residue. The remaining 35% of the structures on the molecule are high mannose type, composed of 5, 6, or 7 mannose residues. Approximately 6% of the high mannose type structures and 10% of the hybrid structures are phosphorylated, as judged by their susceptibility to treatment with alkaline phosphatase. Compositional analysis of an unlabeled preparation of the protein suggested the presence of approximately 1.4 oligosaccharide units per molecule.     

Purification of mouse Ren 2 prorenin produced in Chinese hamster ovary cells

Renin is produced from a larger, inactive precursor, prorenin, by endoproteolytic removal of the amino-terminal prosegment. In this study, we have transfected Chinese hamster ovary cells with the expression plasmid of mouse Ren 2 preprorenin, and have purified mouse Ren 2 prorenin from the incubation medium of these cells by DEAE-Toyopearl chromatography, Blue-Toyopearl chromatography, and isoelectric focusing. Prorenin thus purified has a molecular mass of 42 kDa as determined by SDS-PAGE and an isoelectric point of 6.5. Amino-terminal sequencing has demonstrated that the purified prorenin has the amino-terminus predicted from the nucleotide sequence of mouse Ren 2 preprorenin cDNA.     

Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells

We describe here the properties of a mutant of Chinese hamster ovary cells that expresses a conditional-lethal mutation affecting dense lysosomes. This mutant, termed V.24.1, is a member of the End4 complementation group of temperature-sensitive mutants selected for resistance to protein toxins (Colbaugh, P. A., C.-Y. Kao, S.-P. Shia, M. Stookey, and R. K. Draper. 1988. Somatic Cell Mol. Genet. 14:499-507). Vesicles present in postnuclear supernatants prepared from V.24.1 cells harvested at the restrictive temperature had a 50% reduction in acidification activity, assessed by the ATP-stimulated accumulation of the dye acridine orange in acidic vesicles. To investigate whether specific populations of vesicles were impaired in acidification, we measured acidification activity in three subcellular fractions prepared from Percoll gradients: one containing endosomal and Golgi markers, one containing buoyant lysosomes, and the third containing dense lysosomes. Activity in dense lysosomes was reduced by 90%, activity in the buoyant lysosome fraction was unaffected, and activity in the endosome-Golgi fraction was mildly reduced. The activity of three lysosomal enzymes--beta-hexosaminidase, beta-galactosidase, and beta-glucocerebrosidase--was also reduced in dense lysosomes but nearly normal in the buoyant lysosome fraction. However, beta-hexosaminidase and beta-glucocerebrosidase activity was increased two- to threefold in the endosome-Golgi fraction. We conclude that the lesion selectively impairs dense lysosomes but has little effect on properties of buoyant lysosomes.     

Expression of biologically active human follitropin in Chinese hamster ovary cells

To study the structure-function relationships of follitropin (FSH), we expressed the hormone in a heterologous cell system. A genomic clone bearing a 3.7-kilobase FSH beta insert containing the entire coding sequence was transfected alone or together with the alpha subunit gene into Chinese hamster ovary cells and stable lines expressing either FSH beta or FSH dimer were selected. Pulse-chase experiments revealed that, when transfected alone FSH beta was very slowly secreted similar to lutropin beta and thyrotropin beta but unlike choriogonadotropin beta which is efficiently secreted. However, cotransfection of the FSH beta and alpha subunit genes resulted in "rescue" of the beta subunit and rapid secretion of dimer. These data support the hypothesis that the glycoprotein hormones of pituitary origin have determinants for secretion that differ from those on the placental hormone, choriogonadotropin. Recombinant FSH stimulated steroidogenesis comparable to purified human FSH isolated from pituitaries in an in vitro rat granulosa cell assay and appears more homogeneous by chromatofocusing. Human FSH produced by this cell line provides a source of bioactive FSH for experimental and clinical use.     

Squalene synthase-deficient mutant of Chinese hamster ovary cells.

Squalene synthase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) converts farnesyl pyrophosphate to squalene, the first metabolic step committed solely to the biosynthesis of sterols. Using a fluorescence-activated cell sorting technique designed to screen for cells defective in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, we isolated a squalene synthase-deficient mutant of Chinese hamster ovary cells. The mutant cell line, designated SSD, exhibits less than 7% of the squalene synthase activity of the parental cell line, CHO-HMGal. Both the SSD and the parental cells stably express HMGal, a model protein for studying the regulated degradation of HMG-CoA reductase, which consists of the membrane domain of HMG-CoA reductase fused to bacterial beta-galactosidase (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). In this study, the regulatory effects of mevalonate and compactin on the activity levels of HMGal are substantially reduced in SSD cells as compared to the parental cell line. In lipid-poor medium, SSD cell growth is arrested. The rate of [3H]acetate incorporation into cholesterol for the mutant SSD cells is less than 2% of the rate for the parental cells. However, the incorporation of [3H] squalene into sterols is essentially wild type for SSD cells. When the mutant SSD cells are fed [3H]acetate, radioactivity accumulates in farnesol, much of which is secreted into the medium. By growing SSD cells in lipid-poor medium, a revertant cell type, designated SSR, was isolated. In every assay performed the revertant SSR cells exhibited a phenotype that was essentially wild type, demonstrating that the SSD mutant phenotype was the result of a single mutation.     

Expression and characterization of human apolipoprotein A-I in Chinese hamster ovary cells

We produced human apolipoprotein A-I (apoA-I) in Chinese hamster ovary (CHO) cells. The CHO cells were transfected with an expression plasmid which placed the human apoA-I gene under the direction of the human metallothionein II gene promoter. Isolation of a clonal cell line resulted in high level expression of apoA-I. Greater than 30% of total protein secreted by these CHO cells was apoA-I, which enabled us to purify apoA-I with a single step purification scheme. As a result, large quantities of apoA-I can be produced and isolated without having to rely on plasma sources. Structural characterization of the recombinant apoA-I showed it to be identical to authentic apoA-I from human serum high density lipoprotein. Furthermore, we demonstrated approximately equal to 90% of the apoA-I secreted by CHO cells is processed, mature protein. A portion of the secreted recombinant apoA-I was associated with lipid and floated at a density approximately equal to 1.10 g/ml. Additional analysis identified the presence of five isoforms of apoA-I in the CHO cell conditioned medium. Processing and post-translational modification of the recombinant apoA-I occurred in the CHO cell cultures in the absence of serum components. We conclude that the human apoA-I produced by CHO cells is identical to circulating, mature apoA-I in humans and that recombinant mammalian expression offers an opportunity to investigate apoA-I processing.     

Production of human mutant biologically active hepatocyte growth factor in Chinese hamster ovary cells

Hepatocyte growth factor (HGF) is a potent multifunctional cytokine that affects proliferation, migration, and morphogenesis of various cells. HGF is secreted as an inactive single-chain precursor protein and activated by the cleavage of serine proteases to form heterodimers. In our current study, the cleavage site of HGF was blocked by replaced Arg 494 of Glu (R494E) that resulted in the single-chain HGF (R494E) unable to be cleaved by serine proteases. We established Chinese hamster ovary (CHO) cells overexpressing HGF (R494E), the expression of HGF (R494E) achieved 12?mg/L and was similar to a previously reported study. The recombinant protein was then purified from culture medium using a two-step chromatographic procedure that resulted in about a 40% recovery rate. The purified HGF (R494E) was obtained as a single-chain active protein. It concluded that HGF (R494E) exhibited a biologically active protein and the overexpressing CHO cell line supplied sufficient material for future studies. The R494E replacement of the cleavage site would be beneficial to the utility of other similar therapeutic proteins.     

Biochemical characterization of a mutant asparaginyl-tRNA synthetase from Chinese hamster ovary cells

The biochemical and physical properties of asparaginyl-tRNA synthetase from wild type Chinese hamster ovary cells and a temperature sensitive mutant strain (lys 65a) are compared. The asparaginyl-tRNA synthetase in the mutant strain exhibits a greater temperature lability in vitro, a higher temperature-independent Km for asparagine, and a lower temperature-dependent catalytic capacity than the enzyme from the wild type strain. The mutant enzyme shows no differences in its molecular weight, its Km for tRNAAsn, or its ability to aminoacylate tRNAAsn isoacceptor species compared to the wild type enzyme. These observations, as well as the growth properties of the mutant cells as a function of temperature and exogenous asparagine concentrations, are consistent with their decreased ability to aminoacylate tRNAAsn in vivo.     

Expression of three human beta-adrenergic-receptor subtypes in transfected Chinese hamster ovary cells.

The genes coding for three pharmacologically distinct subtypes of human beta-adrenergic receptors (beta 1 AR, beta 2 AR and beta 3 AR) were transfected for expression in Chinese hamster ovary (CHO) cells. Stable cell lines expressing each receptor were analyzed by ligand binding, adenylate cyclase activation and photoaffinity labeling, and compared to beta AR subtypes observed in previously described tissues, primary cultures and transfected cell lines. Each of the three receptor subtypes displayed saturable [125I]iodocyanopindolol-binding activity. They showed the characteristic rank order of potencies for five agonists, determined by measuring the accumulation of intracellular cAMP. These recombinant cell lines express a homogeneous population of receptors and display the known pharmacological properties of beta 1 AR and beta 2 AR, in human tissues. It is therefore likely that the pattern of ligand binding and adenylate cyclase activation, mediated by the new beta 3 AR in CHO cells, also reflects the yet-undetermined pharmacological profile in humans.     

Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells

Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 microg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 microM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future.     

Cycloheximide-resistance in Chinese hamster ovary cells and human fibroblast cells

Summary A total of 3000 men living in Yamaguchi were screened for glucose-6-phosphate dehydrogenase (G6PD) deficiency using Beutler's spot test and three types of starch gel electrophoresis. These electrophoresis used a phosphate buffer system at pH 7.0, a TRIS-EDTA-borate buffer system at pH 8.6, and a TRIS-hydrochloride buffer system at pH 8.8. Fifteen G6PD-deficient variants were found at the rate of 0.5% and classified into four groups. As new variants, G6PD Konan, Kamiube, and Kiwa were identified. These three variants had a mild to moderate G6PD deficiency and were not associated with any clinical signs. G6PD Konan had fast electrophoretic mobility as compared with normal levels, G6PD Kiwa had slightly elevated electrophoretic mobility, and G6PD Kamiube had normal electrophoretic mobility. These three variants had normal levels of Km G6P, Km NADP, and Ki NADPH, normal utilizations of both 2-deoxy-G6P and deamino-NAPD, normal heat stability, and a normal pH curve. The other variant was G6PD Ube, which we had previously found in Yamaguchi (Nakashima et al., 1977). One boy with G6PD Ube was Korean.     

Expression and purification of recombinant human Angiopoietin-1 produced in Chinese hamster ovary cells

Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation. In an effort to produce large quantities of Ang1, recombinant Chinese hamster ovary (rCHO) cells expressing a high level of recombinant human Ang1 protein (rhAng1) with an amino terminal FLAG-tag were constructed by transfecting the expression vector into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in a medium containing step-wise increments of methotrexate, such as 0.02, 0.08, and 0.32 μM. The rhAng1 secreted from rCHO cells was purified at a purification yield of 18.4% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng1 as heterogeneous multimers. Moreover, rhAng1 expressed in rCHO cells is biologically active in vitro as demonstrated by its ability to bind to the Tie2 receptor and to phosphorylate Tie2. Therefore, the rhAng1 produced from CHO cells could be useful for clarifying the biological effects of exogenous rhAng1 in the future.     

Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells

Acidification of endocytic compartments is necessary for the proper sorting and processing of many ligands and their receptors. Robbins and co-workers have obtained Chinese hamster ovary (CHO) cell mutants that are pleiotropically defective in endocytosis and deficient in ATP- dependent acidification of endosomes isolated by density centrifugation (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308). In this and the following paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2723-2733) we describe detailed studies of endosome acidification in the mutant and wild-type CHO cells. Here we describe a new microspectrofluorometry method based on changes in fluorescein fluorescence when all cellular compartments are equilibrated to the same pH value. Using this method we measured the pH of endocytic compartments during the first minutes of endocytosis. We found in wild- type CHO cells that after 3 min, fluorescein-labeled dextran (F-Dex) was in endosomes having an average pH of 6.3. By 10 min, both F-Dex and fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) had reached acidic endosomes having an average pH of 6.0 or below. In contrast, endosome acidification in the CHO mutants DTG 1-5-4 and DTF 1-5-1 was markedly slowed. The average endosomal pH after 5 min was 6.7 in both mutant cell lines. At least 15 min was required for F-Dex and F-alpha 2M to reach an average pH of 6.0 in DTG 1-5-4. Acidification of early endocytic compartments is defective in the CHO mutants DTG 1-5-4 and DTF 1-5-1, but pH regulation of later compartments on both the recycling pathway and lysosomal pathway is nearly normal. The properties of the mutant cells suggest that proper functioning of pH regulatory mechanisms in early endocytic compartments is critical for many pH-mediated processes of endocytosis.     

Structure of mutant alleles at the aprt locus of Chinese hamster ovary cells

To determine the types of gene structural alterations causing deficiency of adenine phosphoribosyl transferase (aprt) activity in spontaneous and chemically induced mutations of cultured somatic cells, we analyzed the restriction enzyme cleavage patterns of aprt gene sequences in mutant strains selected from Chinese hamster ovary cells. Patterns of aprt-containing fragments in Southern blots were mostly unchanged in our collection of 280 ethyl methane sulfonate-induced and spontaneous aprt- mutants, suggesting that base-pair changes or other alterations below our limit of resolution on agarose gels (approximately 50 base-pairs) are responsible for the great majority of mutations at the aprt locus. Occasionally, these mutations could be localized when they resulted in the loss or gain of a restriction enzyme site and the generation of new fragments of predictable size. Deletions of aprt-containing sequences were detected in only eight of 119 spontaneous mutants and in only one ethyl methane sulfonate-induced mutant. An insertion of 300 base-pairs near the 5' end of the aprt structural gene was found in one spontaneous aprt- strain. This insertion mutant was stable with a reversion frequency of less than 2 X 10(-7). Several unstable aprt- mutants were detected in our collection, but these had no observable alterations of aprt coding or flanking sequences.     

Expression of recombinant human choriogonadotropin in Chinese hamster ovary glycosylation mutants

Human CG, a member of the glycoprotein hormone family that includes LH, FSH, and TSH, is composed of two nonidentical subunits each containing two asparagine linked (N-linked) oligosaccharides. The role of the oligosaccharides in the action of these hormones is unclear. To examine the structure-activity relationships of the glycoprotein hormone oligosaccharides using nonenzymatic and nonchemical methods, we transfected CG subunit genes into mutant cell lines derived from Chinese hamster ovary cells. Two mutant cell lines that synthesize truncated oligosaccharides were used. Cell line 15B, lacking N-acetylglucosaminyltransferase I, synthesizes N-linked carbohydrates containing Man5 oligomannosyl structures, and 1021, defective in transporting CMP-sialic acid into the Golgi, results in sialic-acid deficient glycoproteins. The binding of these derivatives to the LH/CG receptor did not differ significantly from purified CG (CR119), but the ability of the mutant hormones to stimulate cAMP biosynthesis in vitro is reduced compared to wild-type CG or CR119. Since the amino acid sequence of CG from the mutant and wild-type cells is identical, these data indicate that oligosaccharide structures, while not influencing receptor binding, directly affect signal transduction.     

Production of human thyroid-stimulating hormone in Chinese hamster ovary cells

Human thyroid-stimulating hormone (hTSH) has been produced in Chinese hamster ovary (CHO) cells co-transformed with two plasmids: one carrying the alpha subunit cDNA with mouse dihydrofolate reductase gene and the other carrying hTSH beta subunit cDNA. Each cDNA was driven to expression under the control of SV40 early promoter. hTSH and its alpha subunit were secreted into culture media, and their secretion increased with exposure of the cells to increasing concentrations of methotrexate. Gel filtration analysis revealed that the molecular size of the hTSH was the same as that of natural hTSH. Furthermore, the CHO cell-produced hTSH elevated the cyclic AMP level in the rat thyroid cell line FRTL-5 in the same manner as natural hTSH does.     

Expression of recombinant rat Neurotrophin-3 in Chinese hamster ovary cells

The CHO cell line stably producing recombinant rat NT-3 was established. The insertion of rNT-3 cDNA into transferred cell gonome was analyzed with Southern blot. The expressed protein was identified by Dot ELISA (enzyme-linked immunosorbent assay) and Western blot. Western blot showed a clear specifie band of about 14 ku for NT-3. The mean level of rNT-3 in four NT-3eDNA/CHO cell lines was about 2 100 ng/10~6 cells/48 h determined by EIA. The conditioned-medium (CM) of NT-3cDNA/CHO cells could promote the fiber outgrowth of the dissociated dorsal root ganglion of 8-day-old chick embryos, which shows a dose-response relationship. A half-maximal concentration of the biological activity (EC50) of the recombinant protein was approximately 16.7 ng/mL. The MoAb 3W3 of NT-3 could neutralize the biological activity of the rNT-3.     

Expression and function of beta-tubulin isotypes in Chinese hamster ovary cells

The availability of isotype-specific antisera for beta-tubulin, coupled with genetic and biochemical analysis, has allowed the determination of beta-tubulin isotype expression and distribution in Chinese hamster ovary (CHO) cells. Using genetic manipulations involving selection for colcemid resistance followed by reversion and reselection for drug resistance, we have succeeded in isolating cell lines that exhibit three major and one minor beta-tubulin spots by two-dimensional gel electrophoresis. In concert with isotype-specific antibodies, analysis of these mutants demonstrates that CHO cells express two copies of isotype I, at least one copy of isotype IV, and very small amounts of isotype V. All three isotypes assemble into both cytoplasmic and spindle microtubules and are similar in their responses to cold, colcemid, and calcium-induced depolymerization. They have comparable turnover rates and are equally sensitive to depression of synthesis upon colchicine treatment. These results suggest that beta-tubulin isotypes are used interchangeably to assemble microtubule structures in CHO cells. However, of 18 colcemid-resistant mutants with a demonstrable alteration in beta-tubulin, all were found to have the alteration in isotype I, thus leaving open the possibility that subtle differences in isotype properties may exist.