Mammalian Expression System Research Articles

Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation

One of the fastest growing fields in the pharmaceutical industry is the market for therapeutic glycoproteins. Today, these molecules play a major role in the treatment of various diseases, and include several protein classes, i.e., clotting factors, hormones, cytokines, antisera, enzymes, enzyme inhibitors, Ig-Fc-Fusion proteins, and monoclonal antibodies. Optimal glycosylation is critical for therapeutic glycoproteins, as glycans can influence their yield, immunogenicity and efficacy, which impact the costs and success of such treatments. While several mammalian cell expression systems currently used can produce therapeutic glycoproteins that are mostly decorated with human-like glycans, they can differ from human glycans by presenting two structures at the terminal and therefore most exposed position. First, natural human N-glycans are lacking the terminal Gal? 1–3Gal (alpha-Gal) modification;

P2-05-02: PGC1, Peroxisome Proliferator Activated Receptor-gamma (PPAR-gamma) Coactivator-1, Is Necessary in PPAR-gamma Modulated Angiogenesis.

Abstract Background. Peroxisome proliferator activated receptor- gamma (PPAR-gamma), a nuclear hormone receptor, has been shown to be an important regulator of gene expression. PPAR-gamma agonists have been shown to have anti-cancer properties by ways of inducing expression of tumour suppressor genes. It has been indicated that this action of PPAR-gamma is manifested by forming a complex with the PPAR-gamma coactivators (PGC1 &2) and in association with other transcription factors such as SRC-1 and CREB binding proteins. Both PPAR-gamma and PGC-1 have been shown to be aberrantly expressed in human cancer including human breast cancer (1). PPAR-gamma also plays a role in regulating angiogenesis and that PPAR-gamma agonists have been shown to have anti-angiogenic functions. In the present study, we investigated the role of PGC-1 in PPAR-gamma mediated actions on vascular endothelial cells. Materials and methods. Mammalian expression systems for human full length PGC-1 ereonstructed. Anti-PGC-1 transgenes were constructed again using human mammalian expression vector in order to knock down PGC-1 transcript from the cells. Human vascular endothelial cell, HECV, was used in the study. Cell functions linked to angiogenesis including cell adhesion and cellular migration was evaluated using the electric cell impedance sensing method. PPAR-gamma agonists and antagonists were used in the cell models during the analyses. Results. HECV cells weakly expressed PGC-1 and PPAR-gamma. Using the transgenes created, sublines over-expressing PGC1 (HECVPGC1exp) or with PGC-1 expression knockdown (HECVPGC1KD) were established. Loss of PGC1 showed marked increase in both adhesion and cellular migration, opposite was true forHECVPGC1exp cells. PPAR-gamma agonist, pioglitizone and ciglitizone, reduced the adhesion of control cells. In contrast, PPAR-gamma antagonist SR202 exhibited a concentration dependent stimulation of both adhesion and migration of the control cells. Knocking down PGC1 in endothelial cells rendered cells lost response to SR202. Conclusions. PGC-1 is essential in during the modulation of endothelial cells by PPAR-gamma. Together with the aberrant expression of both PPAR-gamma and PGC-1 in breast cancer, it is concluded that PGC-1 is a central player in PPAR-gamma mediated action in both cancer cells and angiogenesis. 1. Jiang WG, Douglas-Jones A, Mansel RE. Expression of peroxisome-proliferator activated receptor-gamma (PPARgamma) and the PPARgamma co-activator, PGC-1, in human breast cancer correlates with clinical outcomes. Int J Cancer. 2003, 106(5):752–757. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-02.

Directed evolution of mammalian anti-apoptosis proteins by somatic hypermutation

Recently, researchers have created novel fluorescent proteins by harnessing the somatic hypermutation ability of B cells. In this study, we examined if this approach could be used to evolve a non-fluorescent protein, namely the anti-apoptosis protein Bcl-x(L), using the Ramos B-cell line. After demonstrating that Ramos cells were capable of mutating a heterologous bcl-x(L) transgene, the cells were exposed to multiple rounds of the chemical apoptosis inducer staurosporine followed by rounds of recovery in fresh medium. The engineered B cells expressing Bcl-x(L) exhibited progressively lower increases in apoptosis activation as measured by caspase-3 activity after successive rounds of selective pressure with staurosporine treatment. Within the B-cell genome, a number of mutated bcl-x(L) transgene variants were identified after three rounds of evolution, including a mutation of Bcl-x(L) Asp29 to either Asn or His, in 8 out of 23 evaluated constructs that represented at least five distinct Ramos subpopulations. Subsequently, Chinese hamster ovary (CHO) cells engineered to overexpress the Bcl-x(L) Asp29Asn variant showed enhanced apoptosis resistance against an orthogonal apoptosis insult, Sindbis virus infection, when compared with cells expressing the wild-type Bcl-x(L) protein. These findings provide, to our knowledge, the first demonstration of evolution of a recombinant mammalian protein in a mammalian expression system.

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Opposite temperature effect on transport activity of KCC2/KCC4 and N(K)CCs in HEK-293 cells

BackgroundCation chloride cotransporters play essential roles in many physiological processes such as volume regulation, transepithelial salt transport and setting the intracellular chloride concentration in neurons. They consist mainly of the inward transporters NCC, NKCC1, and NKCC2, and the outward transporters KCC1 to KCC4. To gain insight into regulatory and structure-function relationships, precise determination of their activity is required. Frequently, these analyses are performed in HEK-293 cells. Recently the activity of the inward transporters NKCC1 and NCC was shown to increase with temperature in these cells. However, the temperature effect on KCCs remains largely unknown.FindingsHere, we determined the temperature effect on KCC2 and KCC4 transport activity in HEK-293 cells. Both transporters demonstrated significantly higher transport activity (2.5 fold for KCC2 and 3.3 fold for KCC4) after pre-incubation at room temperature compared to 37°C.ConclusionsThese data identify a reciprocal temperature dependence of cation chloride inward and outward cotransporters in HEK-293 cells. Thus, lower temperature should be used for functional characterization of KCC2 and KCC4 and higher temperatures for N(K)CCs in heterologous mammalian expression systems. Furthermore, if this reciprocal effect also applies to neurons, the action of inhibitory neurotransmitters might be more affected by changes in temperature than previously thought.

Next-generation sequencing of the CHO cell transcriptome

Since 1957 Chinese hamster ovary (CHO) cells are used for in vitro cultivation as they require assimilable low sustenance [1]. Today, CHO cell lines represent the most commonly used mammalian expression system for the production of therapeutic proteins and are considered as the mammalian equivalent of E. coli in research and biotechnology [2]. The production of biopharmaceuticals in CHO cells is superior to protein production in bacteria, because mammalian cell lines procure complex folding and post-translational modifications like glycosylation. However, contrary to the increasing importance in biotechnology and industry, comprehensive genome and transcriptome information of CHO cell lines is still rare. In this study, the pyrosequencing technology from 454 Life Sciences and a novel assembly approach for cDNA sequences were used to achieve a major step forward towards unraveling the transcriptome of CHO cells. CHO cDNA samples derived from different CHO cell lines and growth conditions were used for the generation of 1.84 mill. high quality sequencing reads with an average read length of 373 nt summing up to 603 Mb data. Assembly of the sequencing data resulted in 41,039 contiguous sequences. These contigs were grouped by the Newbler software into 36,383 isotigs and 28,039 isogroups. Taxonomical classification and comparison to the Mus musculus transcriptome demonstrated the actual quality of the CHO cell line sequences. Metabolic pathways of the central carbohydrate metabolism and biosynthesis routes of sugars used for protein N-glycosylation were reconstructed from the transcriptome data. All relevant genes representing major steps in the N-glycosylation pathway and the central metabolism of CHO cells were detected. Only fructose-1,6-bisphosphatase (3.1.3.11) and 6-phosphogluconolactonase (3.1.1.31) were not identified within the pentose phosphate pathway. The newly sequenced CHO cell line transcriptome was the basis for the design of a customized CHO microarray. Contig sequences were used for the design of 94.580 probes. The designed probes cover 31,905 splice variants of CHO transcripts. With a Self-Self Hybridization experiment (Figure ​(Figure1)1) the functionality of the probes was demonstrated. This experiment was performed with the same RNA as which was used for sequencing. Half of this RNA was labeled with Cy3, the other half with Cy5. For this study the dye intensity, the dye ratio and the adjusted p-value (student´s t-test, FDR controlled, α=0.05) of four microarray replicates were analyzed. It is expected, that the two labeled RNA samples bind equally to the probe, if a transcript is expressed (Figure ​(Figure1:1: pink, dark blue, red, light blue). Only the probes for one transcript could be rejected, because of their dysfunctionality (Figure ​(Figure1,1, green). Figure 1 Self-Self Hybridization experiment of the customized CHO microarray. The dye ratio (M-value) is plotted against the adjusted p-value (student´s t-test, FDR controlled, α=0.05). The dye ratio was calculated using the mean intensity values ... This CHO microarray is now available for further experiments and will support transcriptional analysis of CHO cells under process conditions for cell line and process optimization. It was used already used successfully for a gene expression study of CHO DP-12 cells cultivated under sodium butyrate treatment [3].

Clinically Significant Aberrant HAS1Vb Splicing of the Hyaluronan Synthase 1 Gene (HAS1) Requires a Combination of Mutations and Deletions in Introns 3 and 4 of HAS1 Minigene,

Abstract 3931Clinically important aberrant splicing of the hyaluronan synthase 1 gene (HAS1) occurs in malignant B cells from multiple myeloma (MM) and Waldenstrom macroglobulimenia (WM) patients but is undetectable in B cells from healthy donors (HD)1. HAS1 splice variants are generated by aberrant alternative splicing (AS) within exons 3, 4 and 5, involving exon 4 skipping (Va), partial intron 4 retention (Vc, Vd) or a combination of both (Vb). Aberrant splicing of HAS1Vb has previously shown to correlate with reduced survival in a cohort of MM patients1. In patients, frequent mutations have been identified in introns 3 and 4, suggesting their roles in AS1. To identify genetic regions involved in splice site selection leading to HAS1Vb expression, we performed in vitro sequence manipulation of introns 3 and 4. We established a mammalian expression system to analyse HAS1 splicing by fusing a minigene extending from exon 3 to exon 5 with the upstream cDNA sequence. In an unaltered sequence of the HAS1 minigene, splicing of HAS1 full length predominated, with trace amount of variants, including the novel HAS1Vd, identified for the first time in transfectants. HAS1Vd shared an alternative acceptor site with HAS1Vb (retained 59 bp of downstream intron 4) but unlike HAS1Vb, retained exon 4. Analysis of peripheral blood mononuclear cells (PBMC) revealed that 9% of healthy donors (n=102) and MM patients (n=93) expressed HAS1Vd. About 2% coexpressed HAS1Vb and Vd. In this cohort of patients, HAS1Vb was found in 20% of unfractionated MM PBMC compared to 5% in HD PBMC, supporting our previous finding that HAS1Vb was restricted to sorted MM B cells1and suggesting that in HD PBMC, non-B cells are responsible for this splicing. While MM PBMCs expressed HAS1Vb>Vd, HD PBMCs and transfectants expressed HAS1Vd>Vb, indicating that splicing directed by the minigene construct was substantively different from that occurring in MM patients. In transfectants, partial deletion of HAS1 intron 4 increased HAS1Vd expression but did not affect HAS1Vb despite the fact that both variants use the same 3’ splice site in intron 4. Proper joining of exons 3, 4 and 5 required a minimum of 172 bp of downstream intron 4 and acceptor site selection may be regulated by sequence between 172 and 84 bp upstream of exon 5. Thus, changes in intron 4 alone were insufficient to promote the splicing pattern observed in patients (i.e., elevated HAS1Vb). We then employed site directed mutagenesis to alter multiple G-rich regions in downstream intron 3, a strategy which enhanced exon 4 skipping as shown by increased HAS1Va expression, but did not lead to HAS1Vb splicing. Minimal manipulation to promote exon 4 skipping included base changes within the 2 tandem G-repeats (8 bp) located 73 bp upstream of exon 4, where splicing enhancer/silencer sequences were also identified. Thus, changes in intron 3 also affected the HAS1 splicing profile but like deletions in intron 4, were insufficient on their own to promote HAS1Vb expression. We next developed expression constructs that combined deletion in intron 4 with mutations in intron 3. We are able to demonstrate that when both introns were co-modified, the splicing pattern shifted towards increased HAS1Vb expression, similar to that observed in malignant cells from MM patients. Our previous work showed that deletions or mutations in the 3’ end of intron 4 are frequent in MM; in silico analysis predicts splicing to form HAS1Vb2. To determine whether intron 3 genetic changes occur in patients, we sequenced intron 3 from genomic DNA of 50 MM PBMC. In 22/50 patients, 18 recurrent mutations unique to MM were identified in intron 3; many were also recurrent in our previous study2. Individual mutations recurred in 2–7 patients. Among these, 17/18 recurrent mutations increased the G-C content of intron 3 and 6/18 created or disrupted G runs in intron 3. This supports the idea that in MM patients, cumulative variations in introns 3 and 4 alter splice site selection, operationally resulting in loss of HAS1Vd and enhanced expression of the clinically relevant HAS1Vb variant. We speculate that individuals who accumulate genetic variations in introns 3 and 4 of HAS1 are predisposed to aberrant splicing of HAS1 which may contribute to development of malignancy in MM and WM. Disclosures:Belch:Celgene: Research Funding; Onyx: Research Funding.

TASK Channel Deletion Reduces Sensitivity to Local Anesthetic-induced Seizures

Local anesthetics (LAs) are typically used for regional anesthesia but can be given systemically to mitigate postoperative pain, supplement general anesthesia, or prevent cardiac arrhythmias. However, systemic application or inadvertent intravenous injection can be associated with substantial toxicity, including seizure induction. The molecular basis for this toxic action remains unclear. We characterized inhibition by different LAs of homomeric and heteromeric K channels containing TASK-1 (K2P3.1, KCNK3) and TASK-3 (K2P9.1, KCNK9) subunits in a mammalian expression system. In addition, we used TASK-1/TASK-3 knockout mice to test the possibility that TASK channels contribute to LA-evoked seizures. LAs inhibited homomeric and heteromeric TASK channels in a range relevant for seizure induction; channels containing TASK-1 subunits were most sensitive and IC₅₀ values indicated a rank order potency of bupivacaine > ropivacaine >> lidocaine. LAs induced tonic-clonic seizures in mice with the same rank order potency, but higher LA doses were required to evoke seizures in TASK knockout mice. For bupivacaine, which produced the longest seizure times, seizure duration was significantly shorter in TASK knockout mice; bupivacaine-induced seizures were associated with an increase in electroencephalogram power at frequencies less than 5 Hz in both wild-type and TASK knockout mice. These data suggest that increased neuronal excitability associated with TASK channel inhibition by LAs contributes to seizure induction. Because all LAs were capable of evoking seizures in TASK channel deleted mice, albeit at higher doses, the results imply that other molecular targets must also be involved in this toxic action.

Polyarginine peptide IND-1 enhances recombinant human bone morphogenetic protein-2 yield in mammalian cells

To improve recombinant human bone morphogenetic protein-2 (rhBMP-2) yield, cell lines stably expressing hBMP2 were cultured in the presence of polyarginine peptide IND-1 and showed up to 6-fold increase in the yield of mature BMP-2. Repeated addition of IND-1 to cell cultures consistently improved BMP-2 yields over 53days without affecting cell growth and viability. Investigation of its mechanism of action showed that IND-1 inhibited pro-protein convertase (PC) activity when incubated with cell lysates. However, when intact cells were cultured with IND-1, no change in cellular PC activity was observed. Furthermore, knockdown of furin (a prototypical member of the PCs) in cells did not affect their BMP-2 yields, suggesting furin/PC inhibition is unlikely the mechanism by which IND-1 enhances BMP-2 yields. IND-1 as a medium additive thus enhances BMP-2 production in mammalian cell expression systems.

Synthesis and expression of recombinant interferon alpha-5 gene in tobacco chloroplasts, a non-edible plant

The production of interferon alpha from microbial to mammalian expression system, have certain precincts in terms of cost, scalability, safety and authenticity. Modern biotechnology exploits transgenic crops to get large quantities of complex proteins in a cost-effective way. In order to overcome several challenges from biosafety point of view, the chloroplast transformation strategy is one of the best approaches since plastids are strictly maternally inherited in most of the cultivated species. In the present study the interferon alpha 5 gene was synthesized by using complex set of oligos. After sequence confirmation of the synthesized gene, the histidine residues along with the thrombin protease site were engineered upstream to the synthetic interferon alpha 5 gene. The recombinant fragment was then tethered with chloroplast light inducible promoter, rbcl followed by sequential cloning to develop chloroplast transformation vector to target the cassette into the inverted repeat region of plastome through two events of homologous recombination. The putative transgenic plants obtained through biolistic delivery method and as a result of antibiotic selection of bombarded leaves, were subjected to different rounds of selection and regeneration for homoplasmicity. The spectinomycin-resistant shoots were analyzed through Polymerase Chain Reaction and Sothern blotting. The expression of introduced synthetic genes was recorded using Enzyme Linked ImmunoSorbant Assay technique. It was experienced that mature leaves contained comparatively high levels of interferon compared to young and senescence leaves.

Expression of opioid and anti-opioid receptors in Chinese hamster ovary cells after exposure to dimethyl sulfoxide

Functional and structural studies on G-protein-coupled receptors (GPCRs) at molecular levels require producing and purifying high levels of receptors, and recombinant mammalian cell expression systems constitute the best systems to obtain receptors resembling those expressed in natural environments. In the course of increasing GPCR expression in Chinese hamster ovary (CHO) cells, we have expressed mu (μ)- and kappa (κ)-opioid receptors and neuropeptide FF 1 and FF 2 receptors (NPFF 1 and NPFF 2, respectively) in dimethyl sulfoxide. This treatment did not modify the affinity ( K d) for any receptor, but a significant increase in functional expression levels was observed for all receptors with the noticeable exception of NPFF 1.

Efficient expression screening of human membrane proteins in transiently transfected Human Embryonic Kidney 293S cells

It is often an immense challenge to overexpress human membrane proteins at levels sufficient for structural studies. The use of Human Embryonic Kidney 293 (HEK 293) cells to express full-length human membrane proteins is becoming increasingly common, since these cells provide a near-native protein folding and lipid environment. Nevertheless, the labor intensiveness and low yields of HEK 293 cells and other mammalian cell expression systems necessitate the screening for suitable expression as early as possible. Here we present our methodology used to generate constructs of human membrane proteins and to rapidly assess their suitability for overexpression using transiently transfected, glycosylation-deficient GnT I–HEK 293 cells (HEK 293S). Constructs, in the presence or absence of a C-terminal enhanced green fluorescence protein (EGFP) molecule, are made in a modular manner, allowing for the rapid generation of several combinations of fusion tags and gene paralogues/orthologues. Solubilization of HEK 293S cells, using a range of detergents, followed by Western blotting is performed to assess relative expression levels and to detect possible degradation products. Fluorescence-detection size exclusion chromatography (FSEC) is employed to assess expression levels and overall homogeneity of the membrane proteins, to rank different constructs for further downstream expression trials. Constructs identified as having high expression are instantly suitable for further downstream large scale transient expression trials and stable cell line generation. The method described is accessible to all laboratory scales and can be completed in approximately 3 weeks.

Strong Activation of ether-à-go-go-Related Gene 1 K+ Channel Isoforms by NS1643 in Human Embryonic Kidney 293 and Chinese Hamster Ovary Cells

Two different mechanisms leading to increased current have been described for the small-molecule human ether-à-go-go-related gene (herg) activator NS1643 [1,3-bis-(2-hydroxy-5-trifluoromethylphenyl)-urea]. On herg1a channels expressed in Xenopus laevis oocytes, it mainly acts via attenuation of inactivation and for rat (r) erg1b channels expressed in human embryonic kidney (HEK)-293 cells, it strongly shifts the activation curve to the left. We now investigated the NS1643 effects on erg1b channels in more detail and performed comparative experiments with rat and human erg1a in different expression systems. Significant differences were observed between expression systems, but not between the rat and human isoform. In HEK-293 or Chinese hamster ovary (CHO) cells, activation of rat erg1b channels occurred in a dose-dependent manner with a maximum current increase of 300% obtained with 10 μM NS1643. In contrast, the NS1643-induced strong leftward shift in the voltage dependence of activation further increased with higher drug concentration, needed more time to develop, and exhibited use dependence. Coexpression of KCNE1 or KCNE2 did not attenuate this NS1643 effect on erg1 channel activation and did thus not mimic the lower drug potency on this parameter observed in oocytes. NS1643 (10 μM) slowed erg1b channel deactivation and recovery from inactivation without significant changes in activation and inactivation kinetics. With the exception of accelerated activation, NS1643 affected erg1a channels similarly, but the effect was less pronounced than in erg1b or erg1a/1b channels. It is noteworthy that rerg1b and herg1a inactivation estimated from fully activated current voltage relationships were unaltered in the continued presence of 10 μM NS1643 in the mammalian expression systems, indicating qualitative differences from NS1643 effects in X. laevis oocytes.

HuCAL PLATINUM, a Synthetic Fab Library Optimized for Sequence Diversity and Superior Performance in Mammalian Expression Systems

This article describes the design of HuCAL ( human combinatorial antibody library) PLATINUM, an optimized, second-generation, synthetic human Fab antibody library with six trinucleotide-randomized complementarity-determining regions (CDRs). Major improvements regarding the optimized antibody library sequence space were implemented. Sequence space optimization is considered a multistep process that includes the analysis of unproductive antibody sequences in order to, for example, avoid motifs such as potential N-glycosylation sites, which are undesirable in antibody production. Gene optimization has been used to improve expression of the antibody master genes in the library context. As a result, full-length IgGs derived from the library show both significant improvements in expression levels and less undesirable glycosylation sites when compared to the previous HuCAL GOLD library. Additionally, in-depth analysis of sequences from public databases revealed that diversity of CDR-H3 is a function of loop length. Based upon this analysis, the relatively uniform diversification strategy used in the CDR-H3s of the previous HuCAL libraries was changed to a length-dependent design, which replicates the natural amino acid distribution of CDR-H3 in the human repertoire. In a side-by-side comparison of HuCAL GOLD and HuCAL PLATINUM, the new library concept led to isolation of about fourfold more unique sequences and to a higher number of high-affinity antibodies. In the majority of HuCAL PLATINUM projects, 100–300 antibodies each having different CDR-H3s are obtained against each antigen. This increased diversity pool has been shown to significantly benefit functional antibody profiling and screening for superior biophysical properties.

Use of the α-mannosidase I inhibitor kifunensine allows the crystallization of apo CTLA-4 homodimer produced in long-term cultures of Chinese hamster ovary cells.

Glycoproteins present problems for structural analysis since they often have to be glycosylated in order to fold correctly and because their chemical and conformational heterogeneity generally inhibits crystallization. It is shown that the α-mannosidase I inhibitor kifunensine, which has previously been used for the purpose of glycoprotein crystallization in short-term (3-5 d) cultures, is apparently stable enough to be used to produce highly endoglycosidase H-sensitive glycoprotein in long-term (3-4 week) cultures of stably transfected Chinese hamster ovary (CHO) cells. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of the extracellular region of the cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) homodimer expressed in long-term CHO cell cultures in the presence of kifunensine revealed that the inhibitor restricted CTLA-4 glycan processing to Man9GlcNAc2 and Man5GlcNAc2 structures. Complex-type glycans were undetectable, suggesting that the inhibitor was active for the entire duration of the cultures. Endoglycosidase treatment of the homodimer yielded protein that readily formed orthorhombic crystals with unit-cell parameters a=43.9, b=51.5, c=102.9 Å and space group P2(1)2(1)2(1) that diffracted to Bragg spacings of 1.8 Å. The results indicate that kifunensine will be effective in most, if not all, transient and long-term mammalian cell-based expression systems.

Systemy ekspresyjne białek cytochromu P450 w badaniach in vitro metabolizmu leków

Cytochrome P450 proteins are the most important enzymes involved in metabolic activation or detoxification of various drugs used in clinical practice. However, some drug metabolism pathways may be responsible for their increased toxicity. New expression systems of cytochrome P450 proteins in mammalian cells, including human, are designed to explore the influence of metabolism on the cellular and molecular mechanisms of action of potential drugs and those used therapeutically. They can also be used to study the effect of tested compounds on activity and expression of metabolizing enzymes. Human tumor cell lines with overexpression of cytochrome P450 isoenzymes are of particular importance, especially in studies of potential chemotherapeutics. The HepG2 cell line, derived from human liver cancer, is the most commonly used in studies on drug metabolism and toxicity. However, due to the low level of metabolizing enzymes in these cells, the Hep3A4 cell line with overexpression of CYP3A4 isoenzyme was developed. The stable overexpression of cytochrome P450 isoenzymes was also obtained in other human cancer cell lines, including hepatoma HepaRG cells, ovarian cancer IGROV-1 cells, colon cancer Caco-2, and LS180 cells. This review describes currently developed bacterial, yeast, insect and mammalian (including human) cytochrome P450 protein expression systems, in terms of their advantages and disadvantages in the context of their suitability for basic research and use on a commercial scale.

Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy.

NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in 15N, 13C, or 2H. The bacterial expression systems currently in use to obtain isotopic enrichment, however, cannot produce a number of eukaryotic proteins, especially those that require post-translational modifications such as N-linked glycosylation for proper folding or activity. Here, we report the use of an adenovirus vector-based mammalian expression system to produce isotopically enriched 15N or 15N/13C samples of an outer domain variant of the HIV-1 gp120 envelope glycoprotein with 15 sites of N-linked glycosylation. Yields for the 15N- and 15N/13C-labeled gp120s after affinity chromatography were 45 and 44 mg/l, respectively, with an average of over 80% isotope incorporation. Recognition of the labeled gp120 by cognate antibodies that recognize complex epitopes showed affinities comparable to the unlabeled protein. NMR spectra, including 1H-15N and 1H-13C HSQCs, 15N-edited NOESY-HSQC, and 3D HNCO, were of high quality, with signal-to-noise consistent with an efficient level of isotope incorporation, and with chemical shift dispersion indicative of a well-folded protein. The exceptional protein yields, good isotope incorporation, and ability to obtain well-folded post-translationally modified proteins make this mammalian system attractive for the production of isotopically enriched eukaryotic proteins for NMR spectroscopy.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-011-9506-4) contains supplementary material, which is available to authorized users.

Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery

Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a ‘tag after stop codon’ system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS–PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels. In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity.

Identification of a novel temperature sensitive promoter in cho cells

BackgroundThe Chinese hamster ovary (CHO) expression system is the leading production platform for manufacturing biopharmaceuticals for the treatment of numerous human diseases. Efforts to optimize the production process also include the genetic construct encoding the therapeutic gene. Here we report about the successful identification of an endogenous highly active gene promoter obtained from CHO cells which shows conditionally inducible gene expression at reduced temperature.ResultsBased on CHO microarray expression data abundantly transcribed genes were selected as potential promoter candidates. The S100a6 (calcyclin) and its flanking regions were identified from a genomic CHO-K1 lambda-phage library. Computational analyses showed a predicted TSS, a TATA-box and several TFBSs within the 1.5 kb region upstream the ATG start signal. Various constructs were investigated for promoter activity at 37°C and 33°C in transient luciferase reporter gene assays. Most constructs showed expression levels even higher than the SV40 control and on average a more than two-fold increase at lower temperature. We identified the core promoter sequence (222 bp) comprising two SP1 sites and could show a further increase in activity by duplication of this minimal sequence.ConclusionsThis novel CHO promoter permits conditionally high-level gene expression. Upon a shift to 33°C, a two to three-fold increase of basal productivity (already higher than SV40 promoter) is achieved. This property is of particular advantage for a process with reduced expression during initial cell growth followed by the production phase at low temperature with a boost in expression. Additionally, production of toxic proteins becomes feasible, since cell metabolism and gene expression do not directly interfere. The CHO S100a6 promoter can be characterized as cold-shock responsive with the potential for improving process performance of mammalian expression systems.

Automation of large scale transient protein expression in mammalian cells

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI− cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative.