Michael J. Betenbaugh

The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

Chinese hamster ovary (CHO)–derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of the assembled scaffolds with 21 chromosomes isolated by microfluidics to identify chromosomal locations of genes. Furthermore, we investigate genes involved in glycosylation, which affect therapeutic protein quality, and viral susceptibility genes, which are relevant to cell engineering and regulatory concerns. Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production.

A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution

Microalgae have the potential to revolutionize biotechnology in a number of areas including nutrition, aquaculture, pharmaceuticals, and biofuels. Although algae have been commercially cultivated for over 50 years, metabolic engineering now seems necessary in order to achieve their full processing capabilities. Recently, the development of a number of transgenic algal strains boasting recombinant protein expression, engineered photosynthesis, and enhanced metabolism encourage the prospects of designer microalgae. Given the vast contributions that these solar-powered, carbon dioxide-sequestering organisms can provide to current global markets and the environment, an intensified focus on microalgal biotechnology is warranted. Ongoing advances in cultivation techniques coupled with genetic manipulation of crucial metabolic networks will further promote microalgae as an attractive platform for the production of numerous high-value compounds.

Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome

Chinese hamster ovary (CHO) cells, first isolated in 1957, are the preferred production host for many therapeutic proteins. Although genetic heterogeneity among CHO cell lines has been well documented, a systematic, nucleotide-resolution characterization of their genotypic differences has been stymied by the lack of a unifying genomic resource for CHO cells. Here we report a 2.4-Gb draft genome sequence of a female Chinese hamster, Cricetulus griseus, harboring 24,044 genes. We also resequenced and analyzed the genomes of six CHO cell lines from the CHO-K1, DG44 and CHO-S lineages. This analysis identified hamster genes missing in different CHO cell lines, and detected >3.7 million single-nucleotide polymorphisms (SNPs), 551,240 indels and 7,063 copy number variations. Many mutations are located in genes with functions relevant to bioprocessing, such as apoptosis. The details of this genetic diversity highlight the value of the hamster genome as the reference upon which CHO cells can be studied and engineered for protein production.

The effect of mixotrophy on microalgal growth, lipid content, and expression levels of three pathway genes in Chlorella sorokiniana

Nannochloropsis oculata CCMP 525, Dunaliella salina FACHB 435, and Chlorella sorokiniana CCTCC M209220 were compared in mixotrophic and photoautotrophic cultures in terms of growth rate, protein, and lipid content. Growth improved in glucose, and the biomass productivities of N. oculata, D. salina, and C. sorokiniana were found to be 1.4-, 2.2- and 4.2-fold that observed photoautotrophically. However, biomass and lipid production decreased at the highest glucose concentrations. Meanwhile, the content of protein and lipid were significantly augmented for mixotrophic conditions at least for some species. C. sorokiniana was found to be well suited for lipid production based on its high biomass production rate and lipid content reaching 51% during mixotrophy. Expression levels of accD (heteromeric acetyl-CoA carboxylase beta subunit), acc1 (homomeric acetyl-CoA carboxylase), rbcL (ribulose 1, 5-bisphosphate carboxylase/oxygenase large subunit) genes in C. sorokiniana were studied by real-time PCR. Increased expression levels of accD reflect the increased lipid content in stationary phase of mixotrophic growth, but expression of the acc1 gene remains low, suggesting that this gene may not be critical to lipid accumulation. Additionally, reduction of expression of the rbcL gene during mixotrophy indicated that utilization of glucose was found to reduce the role of this gene and photosynthesis.

Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines

In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N-acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N-acetylglucosaminidase that removes a terminal N-acetylglucosamine from the N-glycan. The innermost N-acetylglucosamine residue attached to asparagine residue is also modified with α(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic enzymes responsible for generating the essential donor sugar nucleotide, CMP-N-acetylneuraminic acid, required for sialylation. Inhibition of N-acetylglucosaminidase has also been applied to alter N-glycan processing in insect cells. This review summarizes current knowledge on N-glycan processing in lepidopteran insect cell lines, and recent progress in glycoengineering lepidopteran insect cells to produce glycoproteins containing complex N-glycans. Published in 2004.

Transcriptome and proteome analysis of Chinese hamster ovary cells under low temperature and butyrate treatment

Recombinant Chinese hamster ovary (CHO) cells selected for high productivity are capable of secreting immunoglobulin G (IgG) molecules at a level that rivals plasma cells in vivo. Following butyrate treatment at 33 degrees C, further increases in productivity are observed. To better understand the mechanisms by which this increased productivity is incurred, the transcriptional response of an antibody-producing cell line undergoing these treatments was investigated using oligo-DNA microarrays. Using distance calculations, more than 900 genes were identified as kinetically differentially expressed between the butyrate-treated 33 degrees C culture and the untreated culture. Furthermore, transcript levels of the heavy and light chain IgG genes increased following treatment. Using stable isotope labeling (SILAC), the secretion rate of IgG was investigated by tracking the decay of the isotope label upon switching to unlabeled medium. Both treated and untreated cultures exhibited very similar IgG secretion kinetics. In contrast, the intracellular IgG content was found to be elevated following treatment. This result suggests that increased productivity under treatment is attributable to elevated cellular secretory capacity, rather than shorter holding times in the secretory pathway. This hypothesis is further supported by the results of gene set enrichment analysis (GSEA), which revealed that elements of the secretory pathway, including Golgi apparatus, cytoskeleton protein binding and small GTPase-mediated signal transduction are enriched and thus may play a role in the increased recombinant protein production observed under butyrate treatment at 33 degrees C.

Part II. Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults.

A number of bioreactor configurations have been developed for the manufacture of products from mammalian cell hosts. Even in the most efficient of these, however, problems such as nutrient exhaustion, growth factor deprivation, and toxin accumulations may arise. Consequently, the current effort focused on the feasibility of overexpressing anti-apoptosis genes in baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells as a means of limiting cell death upon exposure to three such insults. Extended periods of glucose deprivation, serum withdrawal, and treatment with ammonium chloride each caused significant damage, often apoptotic in nature, to BHK and CHO cells, typically rendering cultures completely nonviable. The overexpression of bcl-2 and bcl-x(L), however, was able to abrogate the cell death in BHK cultures, though to varying degrees. For instance, the presence of Bcl-2, which did little to suppress apoptosis upon glucose deprivation, significantly improved the viabilities of these cells during serum withdrawal. In contrast, bcl-x(L) overexpression provided BHK cells with enhanced protection in the absence of glucose, allowing cultures to remain viable throughout the entire three week study. CHO cultures, on the other hand, displayed similar trends in survival in response to both glucose and serum deprivation. During these studies, Bcl-x(L) was consistently able to afford cells the highest degree of protection, though Bcl-2 also enhanced culture viabilities and viable numbers. Death suppression following exposure to 50 mM ammonium chloride was observed to a limited extent in both BHK and CHO cells overexpressing bcl-2 and bcl-x(L). However, even during such harsh treatment, Bcl-x(L) was able to enhance the survival of both cultures, providing CHO cells with viable numbers that were nearly 20-fold that of the controls after five days of exposure. Furthermore, the extensions in cell survival provided by the anti-apoptosis gene products enabled the recovery of many of the cultures during rescue attempts in which the death-inducing stimulus was removed. Clearly, engineering cells to better withstand and recover from the insults common during the large scale cultivation of mammalian cells has a number of potential applications in the biopharmaceutical industries where cell death can limit culture productivities.

Proteomic Analysis of Chinese Hamster Ovary Cells

To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using the CHO genome exclusively, which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. Five-hundred four of the detected proteins included N-acetylation modifications, and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions.

An In Vitro Uniaxial Stretch Model for Axonal Injury

AbstractWe have developed a unique uniaxial stretching device to study axonal injury and neural cell death resulting from brain tissue deformations common in traumatic head injuries. Using displacement control rather than force control, this device is capable of achieving strains >70% and strain rates up to 90 s-1, well above those currently used for studying axonal injury. We have demonstrated that the deformation of the specimen was uniaxial, uniform and highly reproducible; the prespecified displacement profiles could be realized almost precisely; and adequate cell adhesion could be achieved readily. The entire device can fit into a biological safety cabinet to maintain sterility, and the specimens are convenient for cell culture. This device can be used to investigate a wide range of biomechanical issues involved in diffuse axonal injury.

Molecular Chaperones Stimulate the Functional Expression of the Cocaine-sensitive Serotonin Transporter

The serotonin transporter (SERT) is anN-glycosylated integral membrane protein that is predicted to contain 12 transmembrane regions. SERT is the major binding site in the brain for antidepressant drugs, and it also binds amphetamines and cocaine. The ability of various molecular chaperones to interact with a tagged version of SERT (Myc-SERT) was investigated using the baculovirus expression system. Overexpression of Myc-SERT using the baculovirus system led to substantial quantities of inactive transporter, together with small amounts of fully active and, therefore, correctly folded molecules. The high levels of inactive Myc-SERT probably arose because folding was rate-limiting due, perhaps, to insufficient molecular chaperones. Therefore, Myc-SERT was co-expressed with the endoplasmic reticulum (ER) molecular chaperones calnexin, calreticulin and immunoglobulin heavy chain binding protein (BiP), and the foldase, ERp57. The expression of functional Myc-SERT, as determined by an inhibitor binding assay, was enhanced nearly 3-fold by co-expressing calnexin, and to a lesser degree on co-expression of calreticulin and BiP. Co-expression of ERp57 did not increase the functional expression of Myc-SERT. A physical interaction between Myc-SERT-calnexin and Myc-SERT-calreticulin was demonstrated by co-immunoprecipitation. These associations were inhibited in vivo by deoxynojirimycin, an inhibitor of N-glycan precusor trimming that is known to prevent the calnexin/calreticulin-N-glycan interaction. Functional expression of the unglycosylated SERT mutant, SERT-QQ, was also increased on co-expression of calnexin, suggesting that the interaction between calnexin and SERT is not entirely dictated by theN-glycan. SERT is the first member of the neurotransmitter transporter family whose folding has been shown to be assisted by the molecular chaperones calnexin, calreticulin, and BiP.