Chinese Hamster Ovary Cell Lysate Research Articles

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α

Chinese hamster ovary (CHO) cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications. However, toxic proteins and membrane proteins are often difficult-to-express in living cells. Alternatively, cell-free protein synthesis can be employed. This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both, cell-based and cell-free approaches. A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis. This resulted in elevated yields, while eliminating the necessity for exogenous additions during cell-free production, thereby substantially enhancing efficiency. Additionally, we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression. These findings provide promising advancements in bioproduction technologies, offering flexibility to switch between cell-free and cell-based protein production as needed.

Comprehensive Glycoproteomic Analysis of Chinese Hamster Ovary Cells.

The Chinese hamster ovary (CHO) cell line is a major expression system for the production of therapeutic proteins, the majority of which are glycoproteins, such as antibodies and erythropoietin (EPO). The characterization glycosylation profile of therapeutic proteins produced from engineered CHO cells and therapeutic functions, as well as side effects, are critical to understand the important roles of glycosylation. In this study, a large scale glycoproteomic workflow was established and applied to CHO-K1 cells expressing EPO. The workflow includes enrichment of intact glycopeptides from CHO-K1 cell lysate and medium using hydrophilic enrichment, fractionation of the obtained intact glycopeptides (IGPs) by basic reversed phase liquid chromatography (bRPLC), analyzing the glycopeptides using LC-MS/MS, and annotating the results by GPQuest 2.0. A total of 10 338 N-linked glycosite-containing IGPs were identified, representing 1162 unique glycosites in 530 glycoproteins, including 71 unique atypical N-linked IGPs on 18 atypical N-glycosylation sequons with an overrepresentation of the N-X-C motifs. Moreover, we compared the glycoproteins from CHO cell lysate with those from medium using the in-depth N-linked glycoproteome data. The obtained large scale glycoproteomic data from intact N-linked glycopeptides in this study is complementary to the genomic, proteomic, and N-linked glycomic data previously reported for CHO cells. Our method has the potential to monitor the production of recombinant therapeutic glycoproteins.

Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates

Antibodies are indispensable tools for basic research as well as diagnostic and therapeutic applications. Consequently, the development of alternative manufacturing strategies which circumvent the hurdles connected to conventional antibody production technologies is of enormous interest. To address this issue, we demonstrate the synthesis of complex antibody formats, in particular immunoglobulin G (IgG) and single-chain variable fragment Fc fusion (scFv-Fc), in a microsome-containing cell-free system based on translationally active chinese hamster ovary (CHO) cell lysates. To mimic the environment for antibody folding and assembly present in living cells, antibody genes were fused to an endoplasmic reticulum (ER)-specific signal sequence. Signal-peptide induced translocation of antibody polypeptide chains into the lumen of ER microsomes was found to be the prerequisite for antibody chain assembly and functionality. In this context, we show the rapid synthesis of antibody molecules in different reaction formats, including batch and continuous-exchange cell-free (CECF) reactions, depending on the amount of protein needed for further analysis. In addition, we demonstrate site-specific and residue-specific labeling of antibodies with fluorescent non-canonical amino acids. In summary, our study describes a novel antibody production platform which combines the highly efficient mammalian protein folding machinery of CHO cells with the benefits of cell-free protein synthesis.

High-yield production of \u201cdifficult-to-express\u201d proteins in a continuous exchange cell-free system based on CHO cell lysates

Cell-free protein synthesis (CFPS) represents a promising technology for efficient protein production targeting especially so called “difficult-to-express” proteins whose synthesis is challenging in conventional in vivo protein production platforms. Chinese hamster ovary (CHO) cells are one of the most prominent and safety approved cell lines for industrial protein production. In this study we demonstrated the ability to produce high yields of various protein types including membrane proteins and single chain variable fragments (scFv) in a continuous exchange cell-free (CECF) system based on CHO cell lysate that contains endogenous microsomal structures. We showed significant improvement of protein yield compared to batch formatted reactions and proved biological activity of synthesized proteins using various analysis technologies. Optimized CECF reaction conditions led to membrane protein yields up to 980 µg/ml, which is the highest protein yield reached in a microsome containing eukaryotic cell-free system presented so far.

Comparison of cell-based versus cell-free mammalian systems for the production of a recombinant human bone morphogenic growth factor.

The human bone morphogenetic protein-2 (hBMP2) is a glycoprotein, which induces de novo bone formation. Here, recombinant production in stably transfected Chinese Hamster Ovary (CHO) cells is compared to transient expression in Human Embryo Kidney (HEK) cells and cell-free synthesis in CHO cell lysates containing microsomal structures as sites of post-translational processing. In case of the stably transfected cells, growth rates and viabilities were similar to those of the parent cells, while entry into the death phase of the culture was delayed. The maximum achievable rhBMP2 concentration in these cultures was 153pg/mL. Up to 280ng/mL could be produced in the transient expression system. In both cases the rhBMP-2 was found to interact with the producer cells, which presumably contributed to the low yields. In the cell-free system, hBMP2 yields could be increased to almost 40μg/mL, reached within three hours. The cell-free system thus approached productivities for the active (renatured) protein previously only recorded for bacterial hosts, while assuring comprehensive post-translational processing.

Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.

Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called “difficult-to-express” proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of “difficult-to-express” proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called “cell-free” protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various “difficult-to-express” proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA.

Cy5 total protein normalization in Western blot analysis

Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence.

VPS33B, a Member of the Sec1/Munc18 Protein Family, Binds to Integrin Beta Subunits and Is Required for αIIbβ3 Outside-in Signaling

Integrins play fundamental roles in many biological processes such as development, immunity, cancer, wound healing, hemostasis, and thrombosis. Integrin activation is essential for cell adhesion, spreading, survival, proliferation, and migration. Integrins are heterodimeric transmembrane glycoproteins composed of a and b subunits. The function of integrins is modulated by bi-directional transmembrane signaling: inside-out and outside-in signaling, which are mediated through the interactions between integrin a or b subunit cytoplasmic tails and intracellular proteins and can be regulated by many different biochemical signaling pathways. αIIbβ3 is a major integrin expressed in megakaryocytes and platelets. Antagonists of αIIbβ3 are potent anti-thrombotic drugs and new inhibitors targeting αIIbβ3 are under preclinical testing or large patient trials to treat acute coronary syndromes. Integrin αIIbβ3 activation involves binding of proteins, including talin, kindlins, Src kinase, and Gα13 to the cytoplasmic domain of its β subunit. To gain insight into αIIbβ3 signaling and to identify new proteins that regulate αIIbβ3 activation, agarose beads conjugated with glutathione S-transferase (GST)-β3 integrin cytoplasmic domain fusion protein (GST-β3CD) or GST-αIIb integrin cytoplasmic domain fusion protein (GST-αIIbCD) were incubated with human platelet lysates. Proteins pulled down by the GST-β3CD or GST-αIIbCD beads were subjected to mass spectrometric analysis. We found that the GST-β3CD but not the GST-αIIbCD beads specifically pulled down a previously unreported protein, the vacuolar protein sorting-associated protein 33B (VPS33B), encoded by the VPS33B gene. To verify that VPS33B is in a complex with αIIbβ3, we infected Chinese hamster ovary (CHO) cells stably expressing αIIbβ3 (A5 cells) with an adenovirus containing FLAG epitope-tagged human VPS33B cDNA. Cells were allowed to adhere and spread on fibrinogen. FLAG-VPS33B exhibited a significant intracellular colocalization with aIIbb3. Immunoblotting analysis revealed a specific association of b3 with precipitated FLAG-VPS33B. The specific interaction was also confirmed by reciprocal immunoprecipitation using a specific β3 antibody. Furthermore, in an in vitro binding assay, we were able to pull down VPS33B from the lysates of CHO cells overexpressing VPS33B by the GST-β3CD beads. The interaction appears to be direct, since we were also able to pull down purified FLAG-VPS33B with GST-β3CD and GST-β1CD proteins. The pulldown assay showed that the β3 fragment spanning residues 716~730 bound efficiently to FLAG-VPS33B. In contrast, the fragments containing either residues 729~762 or residues 744~762 fragments failed to do the same. More importantly, using the rabbit polyclonal antibody against VPS33B, we were able to coimmunoprecipitate endogenous b3 subunit with VPS33B from human platelet lysate. Thus, we identified a novel binding protein of integrin β3.Next, we investigated the role of VPS33B in integrin activation using a recombinant integrin activation model of CHO cells. Overexpression of VPS33B in CHO cells expressing αIIbβ3 (A5 cells) markedly potentiated cell spreading on fibrinogen and F-actin formation. To establish a role of VPS33B in integrin activation in platelets, we created a mouse model with megakaryocyte- and platelet-specific deletion of VPS33B. Platelets lacking VPS33B were defective in spreading on fibrinogen. VPS33B-/- platelets failed to support clot retraction. On the other hand, thrombin-induced fibrinogen binding to platelets and platelet aggregation were not affected by the loss of VPS33B. Collectively, these results demonstrate an essential role of VPS33B in αIIbβ3 outside-in signaling but is not requried for integrin inside-out signaling. We further demonstrate that VPS33B promotes αIIbβ3 outside-in signaling through RhoA and Rac1 activation, leading to clot retraction and cell spreading, respectively. Therefore, our results for the first time establish vesicle trafficking proteins as an important novel class of modifiers of integrin function in platelets and cells. DisclosuresNo relevant conflicts of interest to declare.

Caveolar Fatty Acids and Acylation of Caveolin-1

PurposeCaveolae are cholesterol and sphingolipids rich subcellular domains on plasma membrane. Caveolae contain a variety of signaling proteins which provide platforms for signaling transduction. In addition to enriched with cholesterol and sphingolipids, caveolae also contain a variety of fatty acids. It has been well-established that acylation of protein plays a pivotal role in subcellular location including targeting to caveolae. However, the fatty acid compositions of caveolae and the type of acylation of caveolar proteins remain largely unknown. In this study, we investigated the fatty acids in caveolae and caveolin-1 bound fatty acids.MethodsCaveolae were isolated from Chinese hamster ovary (CHO) cells. The caveolar fatty acids were extracted with Folch reagent, methyl esterificated with BF3, and analyzed by gas chromatograph-mass spectrometer (GC/MS). The caveolin-1bound fatty acids were immunoprecipitated by anti-caveolin-1 IgG and analyzed with GC/MS.ResultsIn contrast to the whole CHO cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids, 0.48 µg palmitic acid, 0.61 µg stearic acid and 0.83 µg oleic acid/caveolae preparation/5×107 cells. Unexpectedly, GC/MS analysis indicated that caveolin-1 was not acylated by myristic acid; instead, it was acylated by palmitic acid and stearic acid.ConclusionCaveolae contained a special set of fatty acids, highly enriched with saturated fatty acids, and caveolin-1 was acylated by palmitic acid and stearic acid. The unique fatty acid compositions of caveolae and acylation of caveolin-1 may be important for caveolae formation and for maintaining the function of caveolae.

4S polycyclic aromatic hydrocarbon receptor (glycine N-methyltransferase) and the aryl hydrocarbon receptor nuclear translocator (hypoxia inducible factor-1β) interaction in Chinese hamster ovary and rat hepatoma cells: 4S PAH-R/ARNT hetero-oligomers?

Rat CYP1A1 promoter-luciferase, transiently transfected wild-type and 4S PAH receptor (glycine N-methyl transferase, GNMT)-transformed Chinese hamster ovary (CHO) cells were exposed to benzo[a]pyrene and assayed for luciferase activity as an indicator of CYP1A1 promoter activity. CHO cells transformed with the rat 4S PAH receptor/GNMT expression vector had twice the induction level of luciferase activity with respect to wild-type CHO cells in concert with previously published reports that the 4S PAH receptor/GNMT mediates benzo[a]pyrene induction of CYP1A1 gene expression. Lysates of GNMT-transformed CHO cells and wild-type H4IIE rat hepatoma cells exposed to benzo[a]pyrene were immuno-precipitated with anti-GNMT antibodies, separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane for Western blot analysis with anti-aryl hydrocarbon receptor nuclear translocator (ARNT, HIF-1β) antibodies. Results of this analysis indicated that the 4S PAH receptor/GNMT forms a hetero-oligomer (dimer?) with ARNT/HIF-1β which dissociates in the presence of B[a]P. These observations further indicate the role of GNMT (which has been shown to be multifunctional) and B[a]P in the induction of CYP1A1 and also a potential role of GNMT in the modulation of hypoxia inducible factor-1 function with respect to the HIF-1β subunit (ARNT).

Hierarchical Interactions of Homeodomain and Forkhead Transcription Factors in Regulating Odontogenic Gene Expression

FoxJ1 is a forkhead transcription factor expressed in multiple tissues during development and a major regulator of cilia development. FoxJ1(-/-) mice present with defects in odontogenesis, and we correlate these defects to hierarchical interactions between homeodomain factors Pitx2 and Dlx2 with FoxJ1 in regulating their expression through direct physical interactions. Chromatin immunoprecipitation assays reveal endogenous Pitx2 and Dlx2 binding to the Dlx2 promoter and Dlx2 binding to the FoxJ1 promoter as well as Dlx2 and FoxJ1 binding to the amelogenin promoter. PITX2 activation of the Dlx2 promoter is attenuated by a direct Dlx2 physical interaction with PITX2. Dlx2 autoregulates its promoter, and Dlx2 transcriptionally activates the downstream gene FoxJ1. Dlx2 and FoxJ1 physically interact and synergistically regulate both Dlx2 and FoxJ1 promoters. Dlx2 and FoxJ1 also activate the amelogenin promoter, and amelogenin is required for enamel formation and late stage tooth development. FoxJ1(-/-) mice maxillary and mandibular incisors are reduced in length and width and have reduced amelogenin expression. FoxJ1(-/-) mice show a reduced and defective ameloblast layer, revealing a biological effect of these transcription factor hierarchies during tooth morphogenesis. These transcriptional mechanisms may contribute to other developmental processes such as neuronal, pituitary, and heart development.

Bone marrow cells produce a novel TSHβ splice variant that is upregulated in the thyroid following systemic virus infection

Although cells of the immune system can produce thyroid-stimulating hormone (TSH), the significance of that remains unclear. Using 5' rapid amplification of cDNA ends (RACE), we show that mouse bone marrow (BM) cells produce a novel in-frame TSHbeta splice variant generated from a portion of intron 4 with all of the coding region of exon 5, but none of exon 4. The TSHbeta splice variant gene was expressed at low levels in the pituitary, but at high levels in the BM and the thyroid, and the protein was secreted from transfected Chinese hamster ovary (CHO) cells. Immunoprecipitation identified an 8 kDa product in lysates of CHO cells transfected with the novel TSHbeta construct, and a 17 kDa product in lysates of CHO cells transfected with the native TSHbeta construct. The splice variant TSHbeta protein elicited a cAMP response from FRTL-5 thyroid follicular cells and a mouse alveolar macrophage (AM) cell line. Expression of the TSHbeta splice variant, but not the native form of TSHbeta, was significantly upregulated in the thyroid during systemic virus infection. These studies characterize the first functional splice variant of TSHbeta, which may contribute to the metabolic regulation during immunological stress, and may offer a new perspective for understanding autoimmune thyroiditis.

SorLA/LR11 Regulates Processing of Amyloid Precursor Protein via Interaction with Adaptors GGA and PACS-1

SorLA has been recognized as a novel sorting receptor that regulates trafficking and processing of the amyloid precursor protein (APP) and that represents a significant risk factor for sporadic Alzheimer disease. Here, we investigated the cellular mechanisms that control intracellular trafficking of sorLA and their relevance for APP processing. We demonstrate that sorLA acts as a retention factor for APP in trans-Golgi compartments/trans-Golgi network, preventing release of the precursor into regular processing pathways. Proper localization and activity of sorLA are dependent on functional interaction with GGA and PACS-1, adaptor proteins involved in protein transport to and from the trans-Golgi network. Aberrant targeting of sorLA to the recycling compartment or the plasma membrane causes faulty APP trafficking and imbalance in non-amyloidogenic and amyloidogenic processing fates. Thus, our findings identified altered routing of sorLA as a major cellular mechanism contributing to abnormal APP processing and enhanced amyloid beta-peptide formation.

Filamin A Binding Stabilizes Nascent Glycoprotein Ibα Trafficking and Thereby Enhances Its Surface Expression

The glycoprotein (Gp) Ib-IX-V complex is essential for platelet-mediated hemostasis and thrombosis. The cytoplasmic domain of its largest polypeptide subunit GpIbalpha possesses a binding region for filamin A, which links GpIb-IX-V to the platelet cytoskeleton. There is evidence that filamin A binding to GpIbalpha directs the surface expression of GpIb-IX. To investigate the mechanism of this effect, we examined GpIbalpha biosynthesis in Chinese hamster ovary (CHO) cells stably co-expressing wild-type or mutant GpIbalpha with GpIbbeta, GpIX with and without filamin A. We observed that surface GpIbalpha expression is enhanced in CHO cells co-expressing human filamin A. In comparison with cells expressing only GpIbalpha, GpIbbeta, and GpIX (CHO-GpIbalpha/betaIX), lysates from CHO-GpIbalpha/betaIX + filamin A-expressing cells showed greater amounts of immature, incompletely O-glycosylated and fully mature GpIbalpha, but lesser amounts of the approximately 15-kDa C-terminal peptide released when the extracellular domain of GpIbalpha is cleaved by proteases. When filamin A binding is eliminated by truncation of GpIbalpha at C-terminal residue 557 or by a deletion between amino acids 560-570, the decreased synthesis of mature GpIbalpha is accompanied by decreased immature GpIbalpha and by an increased immunodetectable C-terminal peptide. The synthesis of mature GpIbalpha in CHO-GpIbalpha/betaIX cells is eliminated by brefeldin A (which inhibits transport out of the endoplasmic reticulum (ER)) and restored by lactacystin (which inhibits proteasomal degradation). These results suggest that GpIbalpha binds to filamin A within the ER and that filamin A binding directs post-ER trafficking of GpIbalpha to the cell surface.

Purification and Characterization of a GDP-fucose:Polypeptide Fucosyltransferase from Chinese Hamster Ovary Cells

A GDP-fucose:polypeptide fucosyltransferase was purified 5000-fold to homogeneity from Chinese hamster ovary cell extracts in the absence of detergent. The purification procedure included two affinity chromatographic steps using the acceptor substrate, a recombinant factor VII EGF-1 domain, and the donor substrate analog, GDP-hexanolamine, as ligands. The purified enzyme migrates as a single band of 44,000 daltons on SDS-polyacrylamide gel electrophoresis and is itself a glycoprotein with more than one high mannose type oligosaccharide chain with a total molecular weight of 4000. The Km values for factor VII EGF-1 domain and GDP-fucose are 15 and 6 microM, respectively. The Vmax is 2.5 micromol.min-1.mg-1. The presence of 50 mM Mn2+ increased the enzyme activity 17-fold, but Mn2+ was not absolutely required, since the enzyme exhibited some activity even in the presence of EDTA. The acceptor substrate specificity was studied using site-directed mutagenesis of human factor IX EGF domain. Only one of several differently folded species could serve as acceptor substrate, although they all had the same molecular weight as determined by liquid chromatography on-line with mass spectrometry. This indicates that the enzyme requires proper folding of the epidermal growth factor domain for its activity.

Control of the .alpha.5.beta.1 integrin/fibronectin interaction in vitro by the serine/threonine protein phosphatase calcineurin

Using Chinese hamster ovary cell lysate, an in vitro assay has been developed to study the interaction of fibronectin with the alpha 5 beta 1 integrin in a cytosolic environment. In our solid phase assay, 96-well microtiter plates were coated with fibronectin in which cell lysate was incubated. A dose-dependent binding of the fibronectin receptor onto the coated plastic was immunodetected by specific polyclonal antibodies raised against the alpha 5 beta 1 integrin. Both soluble fibronectin and PB1, a monoclonal antibody raised against the fibronectin receptor, competed with the alpha 5 beta 1 integrin for binding to the fibronectin-coated plastic. General phosphatase inhibitors used during cell lysis completely abolished the fibronectin/integrin interaction in the assay, indicating that the affinity of the fibronectin receptor might be modulated by a protein phosphatase activity. Furthermore, in this assay, the interaction between the fibronectin receptor and its substrate in a cytosolic environment required intracellular calcium. Additionally, the action of more specific phosphatase inhibitors and the inhibition of the integrin/fibronectin interaction by a monoclonal antibody raised against the calcium/calmodulin-dependent protein phosphatase calcineurin suggested that calcineurin allowed the interaction between the alpha 5 beta 1 integrin and fibronectin. Metabolical labeling experiments showed that alpha 5 beta 1 itself was not the target of phosphorylation/dephosphorylation cascades involving calcineurin and leading to the modulation of integrin affinity. Taken together, these results showed that in vitro one substrate of the serine/threonine protein phosphatase calcineurin regulates the alpha 5 beta 1 integrin affinity by interacting with a yet unidentified effector.

Glycosidase activities of the 293 and NS0 cell lines, and of an antibody‐producing hybridoma cell line

We have previously demonstrated that Chinese hamster ovary (CHO) cell lysates harbor sialidase, beta-galactosidase, beta-hexosaminidase, and fucosidase activities that can accumulate extracellularly in CHO cell culture, thereby potentially leading to extracellular modification of glycoprotein oligosaccharides. The sialidase activity in CHO cell lysates was surprisingly active and stable at pH 7.5, with a half-life of 57 h at 37 degrees C.We have extended this work to determine whether 293, NS0, or hybridoma cell lysates contain similar glycosidase activities. The pH-activity profiles of beta-galactosidase and beta-hexosaminidase in lysates of these three cell lines resemble the pH-activity profiles for these enzymes in CHO cell lysate, whereas the pH-activity profiles of sialidase and fucosidase appear to be cell-type dependent. Sialidase activities were relatively stable at pH 4.5 in 293, NS0, and hybridoma cell lysates. However, the activities in 293 and NS0 cell lysates were unstable at pH 7.5, with no activity remaining after a 2-h incubation at 37 degrees C. The sialidase activity in hybridoma cell lysate was moderately stable at pH 7.5 with 30% of the activity remaining after a 2-h incubation at 37 degrees C. We conclude that the sialidase activites from 293, NS0, and hybridoma cells have characteristics similar to the vast majority of reported mammalian sialidase activities, and that these activities are markedly differant from the CHO cell sialidase activity.Finally, sialidase, beta-galactosidase, beta-hexosaminidase, and fucosidase activities were measured at pH 7 in cell-free bioreactor supernatants of the hybridoma cell line. As previously observed in CHO cell culture, all four glycosidase activities were present in the hybridoma supernatants. However, the sialidase activity in hybridoma supernatant was an order of magnitude lower than in CHO cell culture supernatant despite the fact that the hybridoma cell lysis rate was an order of magnitude higher. (c) 1994 John Wiley & Sons, Inc.

Glycosidase activities in Chinese hamster ovary cell lysate and cell culture supernatant.

To probe the potential for extracellular degradation of glycoprotein oligosaccharides in conjunction with Chinese hamster ovary (CHO) cell culture, an initial characterization of several CHO cell glycosidases was performed using 4-methylumbelliferyl substrates. CHO cell lysates contained sialidase, beta-galactosidase, beta-hexosaminidase, and fucosidase activities with pH optimums near 5.5, 4, 6, and 6.5, respectively. These glycosidase activities were also present in cell-free supernatant samples from commercial CHO cell cultures. The sialidase activity was further characterized. In contrast to previous reports concerning mammalian sialidases, the sialidase activity in CHO cell lysate retained considerable activity at pH 7 and was very stable, with a half-life of 57 h at 37 degrees C. Both the Km and Vmax of CHO lysate sialidase for 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4MU-NeuAc) varied with pH, and this activity was competitively inhibited by 2,3-dehydro-2-deoxy-N-acetylneuraminic acid and by free N-acetylneuraminic acid. The kinetic characteristics and pH-activity profiles of the CHO cell lysate and cell culture supernatant sialidase activities were essentially identical, and both released sialic acid from the glycoprotein fetuin at pH 7.5. These results suggest that the oligosaccharides of glycoproteins secreted by CHO cells can potentially be modified extracellularly by sialidase under culture conditions which promote the release and extracellular accumulation of this enzyme.