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.     

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 and characterization of recombinant human alpha-N-acetylglucosaminidase secreted by Chinese hamster ovary cells

alpha-N-Acetylglucosaminidase (EC 3.2.1.50) is a lysosomal enzyme that is deficient in the genetic disorder Sanfilippo syndrome type B. To study the human enzyme, we expressed its cDNA in Lec1 mutant Chinese hamster ovary (CHO) cells, which do not synthesize complex oligosaccharides. The enzyme was purified to apparent homogeneity from culture medium by chromatography on concanavalin A-Sepharose, Poros 20-heparin, and aminooctyl-agarose. The purified enzyme migrated as a single band of 83 kDa on SDS-PAGE and as two peaks corresponding to monomeric and dimeric forms on Sephacryl-300. It had an apparent K(m) of 0.22 mM toward 4-methylumbelliferyl-alpha-N-acetylglucosaminide and was competitively inhibited by two potential transition analogs, 2-acetamido-1,2-dideoxynojirimycin (K(i) = 0.45 microM) and 6-acetamido-6-deoxycastanospermine (K(i) = 0.087 microM). Activity was also inhibited by mercurials but not by N-ethylmaleimide or iodoacetamide, suggesting the presence of essential sulfhydryl residues that are buried. The purified enzyme preparation corrected the abnormal [(35)S]glycosaminoglycan catabolism of Sanfilippo B fibroblasts in a mannose 6-phosphate-inhibitable manner, but its effectiveness was surprisingly low. Metabolic labeling experiments showed that the recombinant alpha-N-acetylglucosaminidase secreted by CHO cells had only a trace of mannose 6-phosphate, probably derived from contaminating endogenous CHO enzyme. This contrasts with the presence of mannose 6-phosphate on naturally occurring alpha-N-acetylglucosaminidase secreted by diploid human fibroblasts and on recombinant human alpha-l-iduronidase secreted by the same CHO cells. Thus contrary to current belief, overexpressing CHO cells do not necessarily secrete recombinant lysosomal enzyme with the mannose 6-phosphate-targeting signal; this finding has implications for the preparation of such enzymes for therapeutic purposes.     

Purification and characterization of recombinant human interleukin 5 expressed in Chinese hamster ovary cells

Recombinant human interleukin 5 (rhIL-5) expressed in Chinese hamster ovary cells was purified and characterized. Molecular heterogeneity was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two major components of Mr around 40,000 were detected under non-reducing conditions. However, under reducing conditions, the Mr of rhIL-5 was determined to be 22,000 and 20,000. After treatment with endoglycosidase F, a band with an apparent Mr of 18,000 was observed. Treatment of rhIL-5 with 2-mercaptoethanol followed by N-ethylmaleimide resulted in its dissociation into a monomeric form. This alkylated rhIL-5 was biologically less active than intact rhIL-5. These results suggest that rhIL-5 exists as a dimer, and that the heterogeneity of rhIL-5 is mainly due to the difference in the content of carbohydrate. Moreover, the formation of disulfide bond(s) might be important for the biological activity of rhIL-5.     

Isolation and characterization of native human renin derived from Chinese hamster ovary cells

Transfection of Chinese hamster ovary (CHO) cells with a plasmid containing the cDNA for human preprorenin has provided cell lines that secrete 15 mg of native prorenin per liter of culture medium. Tryptic activation of the prorenin occurs by selective cleavage of the Arg66-Leu67 bond (numbering as in preprorenin). The renin product, purified in a single step and in high yield by affinity chromatography, is fully stable for as long as 8 months when stored in solution at 4 degrees C and pH 6.5. Purity of the renin was judged to be greater than 95% by gel electrophoresis, compositional and N-terminal sequence analyses, and specific enzyme activity. An important aspect of the present work is the development of a direct assay for renin which permits accurate and reproducible evaluation of enzyme units and kinetic parameters. Application of methods described herein, combined with appropriate scale-up fermentation capabilities, provides the means for generating gram quantities of human renin and its zymogen.     

Expression, purification, and characterization of human recombinant thrombopoietin in Chinese hamster ovary cells

Thrombopoietin (TPO) is a primary regulator of megakaryocytopoiesis, a process through which megakaryocytes proliferate and mature into platelets. Recombinant human TPO (rhTPO) was expressed in Chinese hamster ovary (CHO) cells and purified from the culture medium. The cDNA encoding full-length TPO, including the native signal peptide sequence, was amplified by PCR from a human fetal liver cDNA library. The product was cloned into a mammalian expression vector under the control of the SV40 early promoter and enhancer. Secreted rhTPO was purified in three conventional chromatography steps. It migrates on SDS-PAGE as a broad band, characteristic of a heavily glycosylated protein, with an average molecular mass of 85 kDa. rhTPO expressed in CHO cells is biologically active in vitro as demonstrated by its ability to stimulate the proliferation of a megakaryocytic cell line and to trigger the JAK/STAT signal transduction pathway. rhTPO also shows activity in vivo as judged by the elevation of platelet count in treated mice.     

Purification and characterization of recombinant human antithrombin containing the prelatent form in Chinese hamster ovary cells

Antithrombin (AT) is a serine proteinase inhibitor and a major regulator of the blood coagulation cascade. AT in human plasma has two isoforms, a predominant alpha-isoform and a minor beta-isoform; the latter lacks N-glycosylation at Asn 135 and has a higher heparin affinity. From the difference in its folding states, the AT molecule can be separated into three forms: a native form, a denatured and inactive form known as the latent form, and a partially denatured form called the prelatent form. In this study, we purified and characterized recombinant human AT (rAT) containing the prelatent form produced by Chinese hamster ovary (CHO) cells. When rAT was purified at physiological pH, its specific activity was lower than that of plasma-derived human AT (pAT). The latent and prelatent forms were detected in rAT by using hydrophobic interaction chromatography analysis. However, when rAT was purified at alkaline pH, the prelatent form was reversibly folded to the native form and the inhibitory activity of rAT increased to a value similar to that of pAT. Highly purified rAT was analyzed and compared with pAT by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, amino acid composition, N-terminal sequence, monosaccharide composition, peptide mapping, and heparin-binding affinity. From these analyses, rAT was found to be structurally identical to pAT, except for carbohydrate side-chains. rAT in CHO cells had a high beta-isoform content and it caused a higher heparin affinity than by pAT and also pH-dependent reversible inhibitory activity.     

Purification and characterization of recombinant human testis angiotensin-converting enzyme expressed in Chinese hamster ovary cells.

Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: p omega-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which full-length and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mg/liter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either p omega-ACE or pLEN-ACE-VII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) approximately 100 kDa. The native and recombinant proteins cross-reacted equally with anti-human kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investigating structure-function relationships in this enzyme.     

Characterization of recombinant human erythropoietin produced in Chinese hamster ovary cells

Physicochemical properties of recombinant human erythropoietin were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human urine when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Sedimentation equilibrium experiments showed the recombinant erythropoietin preparation to be essentially a single macromolecular component with a molecular weight of 30,400 and a carbohydrate content of 39%. The Stokes radius of recombinant erythropoietin was estimated to be 32 A from gel filtration, much larger than the 20-A radius calculated for a sphere of the observed molecular weight. This difference may be ascribed to the extensive glycosylation. The fluorescence and phosphorescence spectra showed that the luminescent tryptophan(s) is (are) solvent-exposed and can be quenched by I- and acrylamide but not by Cs+. On acid titration, the recombinant erythropoietin showed a conformational transition with a midpoint of pH 4.1. This suggests that the net charges on the protein moiety rather than on the whole molecule play a role in protein structure stability.     

Isolation and characterization of nitrogen mustard-sensitive mutants of Chinese hamster ovary cells

Three nitrogen mustard-sensitive lines of Chinese hamster ovary cells were isolated from mutagenized cultures using the procedure of Thompson et al. (1980). The lines, designated NM1, NM2 and NM3, were 2.1-, 17- and 6.8-fold more sensitive to nitrogen mustard, respectively, than their parent, wild-type, line as determined by the dose required to kill 90% of the cells, IC90. Patterns of cross-sensitivity to other DNA-damaging agents including ultraviolet light, cis-diamminedichloroplatinum, and other alkylating agents were determined for each line. Analysis of these results suggests that the phenotypes of the mutant lines are different from those lines reported previously.     

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.     

Expression of a deletion mutant of the prosegment of human prorenin in Chinese hamster ovary cells

Expression plasmids encoding native human preporenin and a mutant deleted in its entire prosegment were transfected into Chinese hamster ovary cells. The cells transfected with the expression plasmid of native preporenin secreted exclusively inactive prorenin, while the cells transfected with the mutant secreted the active enzyme. The secreted amount of renin from the latter cells was much lower than that of prorenin from the former ones, although these two enzymes had little difference in specific activity after trypsin activation. These results suggest that the prosegment plays an important role in the secretory process of renin, although the fully active enzyme can be formed in its absence.     

Isolation and preliminary characterization of bleomycin-resistant mutants from Chinese hamster ovary cells

Stable mutants resistant to an anticancer antibiotic, bleomycin-A2, were selected in Chinese hamster ovary (CHO) cell either spontaneously or after ethylmethane sulfonate mutagenesis. Fluctuation analysis showed that bleomycin resistance occurs in CHO at a rate of 6.50--6.58 x 10(-7) mutations per cell per generation. Bleomycin-A2-resistant cell lines exhibited increased resistance to bleomycin analogs--bleomycin-A5, -B2, -B4, and pepleomycin. Colchicine, mitomycin C, and ultraviolet light irradiation inhibited colony formation equally in CHO cells and in bleomycin-resistant mutants. Cell-cell hybridization tests showed that bleomycin-resistance behaves as a dominant trait. Bleomycin-inactivating activity in the mutant cell extracts was three to fourfold higher than that in extracts of the parental CHO cell.     

Isolation and characterization of tunicamycin resistant mutants from Chinese hamster ovary cells

Stable clones selected for resistance to tunicamycin (TM) have been isolated from Chinese Hamster Ovary (CHO) cells. The TMR phenotype is stable for more than nine months in the absence of the drug. The morphology of TMR mutant varies from epitheloid to abnormally elongate. The mutants do not display cross-resistance for ConA but are slightly cross-resistant to PHA. Biochemically labeled membrane proteins and glycoprotein of Vesicular stomatitis virus (VSV) grown in the TMR mutants revealed that the incorporation of radioactive glucosamine was markedly reduced in the mutants. The results indicate that TMR cells are a novel type of membrane mutant.     

Characterization of recombinant human antithrombin III synthesized in Chinese hamster ovary cells

Biochemical and physiochemical properties of recombinant human antithrombin III were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human plasma when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Comparison of the NH2-terminal sequences of recombinant and human plasma-derived antithrombin III showed that on synthesis and secretion of the recombinant protein from Chinese hamster ovary cells the signal peptide is correctly cleaved by the corresponding endoplasmic signal peptidase. The recombinant antithrombin III has identical properties in heparin binding and biological activities as determined in vitro by two-dimensional immunoelectrophoresis, progressive inhibitor, and heparin cofactor assays. Analysis of the carbohydrate portion of recombinant antithrombin III synthesized in Chinese hamster ovary cells revealed glycosylation of the complex type. Characterization of the oligosaccharide chains present in the recombinant protein reveals three major fractions, A (20%), B (60%), and C (20%). Fraction A contains tri- and tetraantennary complex-type oligosaccharides, fraction B contains biantennary oligosaccharides, and fraction C partially truncated biantennary structures. Pharmacokinetic studies with recombinant and plasma-derived antithrombin III in rabbits showed that the clearance behavior of both proteins is very similar and can be described by a double exponential decrease with almost identical kinetic parameters.     

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.     

Structure of recombinant human interleukin 5 produced by Chinese hamster ovary cells

The complete peptide map of purified recombinant human interleukin 5 (rhIL-5) was determined to verify its primary structure, glycosylation sites, and disulfide bonding structure. Each peptide fragment generated by Achromobacter protease I (API) digestion was purified and characterized by amino acid analysis and amino acid sequence analysis. After digestion with API, we could identify all the peptides which were expected from human IL-5 cDNA sequence. The analyses of sulfhydryl content in rhIL-5 molecule and disulfide-containing peptide obtained from API digestion indicated that active form of rhIL-5 existed as an antiparallel dimer linked by two pairs of Cys-44 and Cys-86. In addition, we concluded that Thr-3 and Asn-28 were glycosylated. The results indicate that primary structure of rhIL-5 is highly homogeneous and observed heterogeneity is due to the difference in the content of carbohydrate.     

Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-gamma

The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc.     

Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human interferon-gamma

A Chinese hamster ovary (CHO) cell line expressing recombinant human interferon-gamma (IFN-gamma) was grown under glucose limitation in a chemostate at a constant dilution rate of 0.015 h(-1) with glucose feed concentrations of 2.75 mM and 4.25 mM. The changes in cell concentration that accompanied changes in the glucose feed concentration indicated that the cells were glucose-limited. The cell yield on glucose remained constant, but there was a decline in residual glucose concentration and a reduced lactate yield from glucose in the latter stages of the culture. The consumption rates for many of the essential amino acids were increased later in the culture. The volumetric rate of interferon-gamma production was maintained throughout the course of this culture, indicating that IFN-gamma expression was stable under these conditions. However, the specific rate of IFN-gamma production was significantly lower at the higher glucose feed concentration. Under glucose limitation, the proportion of fully glycosylated IFN-gamma produced by these cells was less than that produced in the early stages of batch cultures. The proportion of fully glycosylated IFN-gamma increased during transient periods of glucose excess, suggesting that the culture environment influences the glycosylation of IFN-gamma.     

Characterization of recombinant human adipocyte-derived leucine aminopeptidase expressed in Chinese hamster ovary cells

Adipocyte-derived leucine aminopeptidase (A-LAP) is a recently identified novel member of the M1 family of zinc-metallopeptidases. Transfection of the A-LAP cDNA into COS-7 cells resulted in the secretion of the enzyme. In this study, recombinant A-LAP was expressed in Chinese hamster ovary cells, purified to homogeneity and its enzymatic properties were characterized. The purified enzyme was active towards a synthetic substrate, L-leucyl-p-nitroanilide, yielding a V(max) of 3.55 micromol/min/mg and a K(m) of 1.28 mM, and was shown to be a monomeric protein with molecular mass of 120 kDa in solution. By monitoring the sequential N-terminal amino acid liberation, it was found that the enzyme hydrolyzes a variety of bioactive peptides, including angiotensin II and kallidin. Immunohistochemical analysis indicated that the enzyme is expressed in the cortex of the human kidney, where tissue kallikrein is localized. Taken together, these results indicate that A-LAP possesses a broad substrate specificity towards naturally occurring peptide hormones and suggest that it plays a role in the regulation of blood pressure through the inactivation of angiotensin II and/or the generation of bradykinin in the kidney.