High‐yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development

Low‐yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline‐O‐glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY‐2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of ”Ser‐Pro“ dipeptide, or (SP)₃₂, to study cell growth and protein secretion, culture scale‐up, and establishment of perfusion cultures for continuous production. The BY‐2 cells accumulated low levels of cell biomass (～7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)₃₂‐tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY‐2 cells cultured in a 5‐L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day^?1, generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures.     

Process development of a recombinant antibody/interleukin-2 fusion protein expressed in protein-free medium by BHK cells

The production, purification and stability of quality (in terms of integrity and glycosylation) of an antibody/interleukin-2 fusion protein with potential application in tumour-targeted therapy expressed in BHK21 cells are described. Consistency of the product throughout time was determined by analysis of glycosylation of the fusion protein using MALDI-TOF mass spectroscopy and HPAEC-PAD combined with product integrity studies by SDS-PAGE and Western blotting. These investigations showed consistent expression in terms of integrity and of three major oligosaccharide structures of the fusion protein after 62 generations. The data obtained at this stage indicated the suitability of the cell line for production purposes. Different approaches for the production of this protein were subsequently carried out. The relative productivity of the recombinant fusion protein and general performance of the cells in two different protein-free medium (PFM) culture systems, continuous chemostat and continuous perfusion using a Centritech centrifuge as a cell retention device, were studied. The results indicate that the chemostat culture resulted in more stable and controllable nutrient environment, which could indicate better product consistency, in accordance with what has been observed under serum-containing conditions, in relation to the perfusion culture. Finally, product obtained from the chemostat culture was analysed and purified. The purification process was optimised with an increase in the overall yield from 38 to 70% being obtained, a significant improvement with important consequences for the implementation of an industrial-scale culture system. In conclusion, it was possible to produce and purify the recombinant antibody/interleukin-2 fusion protein assuring the quality and stability of the product in terms of integrity and glycosylation. Therefore, a candidate production process was established.     

Bioprocess development for the production of a recombinant MUC1 fusion protein expressed by CHO-K1 cells in protein-free medium

The mucin MUC1 is a candidate for use in specific immunotherapy against breast cancer, but this requires the large-scale production of a MUC1 antigen. In this study, a bioprocess for the expression of a recombinant MUC1 fusion protein with a cancer associated glycosylation in CHO-K1 cells has been developed. Cells permanently expressing parts of the extracellular portion of MUC1 fused to IgG Fc were directly transferred from adherent growth in serum-containing medium to suspension culture in the protein-free ProCHO4-CDM culture medium. Using the Cellferm-pro system, optimal culture parameter as pH and pO(2) were determined in parallel spinner flask batch cultures. A pH of 6.8-7.0 and a pO(2) of 40% of air saturation was found to give best cell growth and productivity of secreted recombinant protein. Specific productivity strongly depended the pO(2) and correlated with the online monitored oxygen uptake rate (OUR) of the cells, which indicates a positive influence of the rate of oxidative phosphorylation on productivity. The optimised conditions were applied to continuous perfusion culture which gave very high cell densities and space time yields of the recombinant MUC1 fusion protein, allowing production at gram scale. The product degradation was much lower in supernatants from continuous perfusion culture compared to batch mode. Antibodies reacting with cancer associated MUC1 glycoforms strongly bound to the fusion protein, indicating that the desired glycoforms were obtained and suggesting that the recombinant MUC1 protein could be tested for use in immunotherapy.     

Product quality of a recombinant fusion protein expressed in immobilised baby hamster kidney cells grown in protein-free medium

Baby hamster kidney cells producing a recombinant IgG-IL2 fusion protein were grown as spinner batch cultures in a protein-free medium, with cells immobilised in porous and non-porous supports at different support concentrations and agitation rates. Product quality, i.e., integrity, decreases up to 60% with the increase in agitation rate or support concentration and from porous to non-porous supports. LDH activity is a good indicator for the monitoring of product degradation, and thus product quality. Optimal conditions regarding titre of intact product were achieved at 60 rpm with 2 g porous support l^–1.     

Expression of a new chimeric protein with a highly repeated sequence in tobacco cells

In wheat, the high-molecular weight (HMW) glutenin subunits are known to contribute to gluten viscoelasticity, and show some similarities to elastomeric animal proteins as elastin. When combining the sequence of a glutenin with that of elastin is a way to create new chimeric functional proteins, which could be expressed in plants. The sequence of a glutenin subunit was modified by the insertion of several hydrophobic and elastic motifs derived from elastin (elastin-like peptide, ELP) into the hydrophilic repetitive domain of the glutenin subunit to create a triblock protein, the objective being to improve the mechanical (elastomeric) properties of this wheat storage protein. In this study, we investigated an expression model system to analyze the expression and trafficking of the wild-type HMW glutenin subunit (GS_W) and an HMW glutenin subunit mutated by the insertion of elastin motifs (GS_M-ELP). For this purpose, a series of constructs was made to express wild-type subunits and subunits mutated by insertion of elastin motifs in fusion with green fluorescent protein (GFP) in tobacco BY-2 cells. Our results showed for the first time the expression of HMW glutenin fused with GFP in tobacco protoplasts. We also expressed and localized the chimeric protein composed of plant glutenin and animal elastin-like peptides (ELP) in BY-2 protoplasts, and demonstrated its presence in protein body-like structures in the endoplasmic reticulum. This work, therefore, provides a basis for heterologous production of the glutenin-ELP triblock protein to characterize its mechanical properties.     

POTE protein, a cancer-testis antigen, is highly expressed in spermatids in human testis and is associated with apoptotic cells

The POTE gene family encodes very closely related proteins that are highly expressed in testis and in many cancers. Recent studies indicate that the POTE proteins have a pro-apoptotic function. To examine if POTE is associated with cells that are undergoing apoptosis in testis, we determined the cellular location of POTE and of Cleaved Caspase-3 in testicular tissues from 26 azoospermic men. We found intense expression of POTE in round spermatids that are undergoing apoptosis, which are positive for Cleaved Caspase-3. This study suggests POTE may have a role in apoptosis in the human testis.     

A new Arabidopsis nucleic-acid-binding protein gene is highly expressed in dividing cells during development

An Arabidopsis thaliana cDNA encoding a new RNA-binding protein (RBP37) was cloned from a silique cDNA library. The predicted amino acid sequence corresponds to a RBP containing two RNA recognition motifs (RRM) and a basic domain. An affinity for nucleic acids was confirmed in binding assays using in vitro synthesised AtRBP37 protein. In situ hybridisation experiments on sections of flowers and siliques showed expression only in growing organs: gynoecium, petals, filaments and during early-embryogenesis expression is located in the embryo proper and the suspensor up to late heart stage. Expression is not detected in the embryo during maturation.This results suggests an expression pattern correlated with dividing cells.     

Improved protein synthesis and secretion through medium enrichment in a stable recombinant yeast strain

Two Saccharomyces cerevisiae strains were employed to investigate the effects of medium enrichment on the expression and secretion of a recombinant protein. One was a stable autoselection strain with mutations in the ura3, fur1, and urid-k genes. The combination of these three mutations blocks both the pyrimidine nucleotide biosynthetic and salvage pathways and is lethal to the cells. Retention of the plasmid, which carries a URA3 gene, was essential for cell viability. Therefore, all media were selective, allowing cultivation of the strain in complex medium. The second strain was a nonautoselection (control) strain and is isogenic to the first except for the fur1 and urid-k mutations. The plasmid utilized contains the yeast invertase gene under the control of the MFalpha1 promoter and leader sequence. The expression and secretion of invertase for the autoselection strain were examined in batch culture for three media: a minimal medium (SD), a semidefined medium (SDC), and a rich complex medium (YPD). Biomass yields and invertase productivity (volumetric activity) increased with the complexity of the medium; total invertase volumetric activity in YPD was 100% higher than in SDC and 180% higher than in SD. Specific activity, however, was lowest in the SDC medium. Secretion efficiency was extremely high in all three media; for the majority of the culture, 80-90% of the invertase was secreted into the periplasmic space and/or culture medium. A glucose pulse at the end of batch culture in YPD facilitated the transport of residual cytoplasmic invertase. For the nonautoselection strain, invertase productivity did not improve as the medium was enriched from SDC to YPD, and plasmid stability in the complex YPD medium dropped from 54% to 34% during one batch fermentation. During long-term sequential batch culture in YPD, invertase activity decreased by 90% and the plasmid-containing fraction dropped from 56% to 8.8% over 44 generations of growth. The expression level for the autoselection strain, however, remained high and constant over this time period, and no reversion at the fur1 or urid-k locus was observed. (c) 1993 John Wiley & Sons, Inc.     

A novel mitogenic protein that is highly expressed in cells of the gastric antrum mucosa

Human and pig cDNAs for a novel stomach protein, the product of a gene expressed at high levels specifically in cells of the antrum mucosa, have been characterized. The general exon/intron structure of the genomic DNA is conserved in humans and mice. The predicted protein sequences of the human and mouse mRNAs contain 185 and 184 amino acids, respectively. The protein isolated from pig antral extracts has an NH2 terminus consistent with cleavage of a 20-amino acid signal peptide. Human cDNA was expressed in E. coli to generate a protein antigen for antibody production. The antibodies detected polypeptides of approximately 18 kDa in antrum extracts from all mammalian species tested. Immunocytochemistry located antrum mucosal protein (AMP)-18 to surface mucosal cells of the mouse antrum and, specifically, to secretion granules, suggesting that it is cosecreted with mucins. Antrum extracts and recombinant human AMP-18 exhibit growth-promoting activity on epithelial cells that can be blocked by the specific antisera. We suggest that AMP-18 is a "gastrokine" that maintains the integrity of the gastric mucosal epithelium.     

Glycine to glutamic acid misincorporation observed in a recombinant protein expressed by Escherichia coli cells

A novel amino acid misincorporation, in which the intended glycine (Gly) residues were replaced by a glutamic acid (Glu), was observed in a recombinant protein expressed by Escherichia coli. The misincorporation was identified by peptide mapping and liquid chromatography-tandem mass spectrometric analysis on proteolyzed peptides of the protein and verified using the corresponding synthetic peptides containing the misincorporated residues. Analysis of the distribution of the misincorporated residues and their codon usage shows strong correlation between this misincorporation and the use of rarely used codon within the E. coli expression system. Results in this study suggest that the usage of the rare codon GGA has resulted in a Glu for Gly misincorporation.     

A novel strategy for production of a highly expressed recombinant protein in an active form.

Under standard growth conditions, E. coli transformed with the high-level expression vector pMON5525 produces recombinant DMAPP/AMP transferase in inactive, insoluble complexes. We have produced large amounts of active, soluble protein by growing and inducing the cells under osmotic stress in the presence of sorbitol and glycyl betaine. This caused an increase of up to 427-fold in the active yield, and the disappearance of the protein from the pelletable fraction of cell extracts. This treatment may have wide applicability.     

Heat-shock protein 90 is associated with microtubules in tobacco cells

Summary The localization of HSP90 (heat-shock protein 90) was analyzed with respect to the microtubular cytoskeleton by double immunofluorescence and confocal laser microscopy in tobacco VBI-O cells during axial cell division and elongation. HSP90 was observed to be colocalized with cortical and radial microtubules and the nuclear envelope in premitotic cells, with the preprophase band, and with the phragmoplast. The HSP90 epitope could not be detected in mature division spindles. The association of the HSP90 epitope with radial and cortical microtubules was not continuous in space. HSP90 was organized in discrete foci that were found to be aligned with microtubules, and the distance between these foci increased, when the cells entered the elongation phase. Elimination of microtubules by drugs resulted in a loss of cell axiality and alignment of the HSP90 epitope. Together with biochemical data demonstrating binding of tobacco HSP90 to tubulin dimers these data indicate a possible role of HSP90 in the organization of microtubules.Abbreviations EPC ethyl-N-phenylcarbamate - FITC fluorescein isothiocyanate - HSP90 heat-shock protein 90 - MAP microtubuleassociated protein - TRITC tetramethylrhodamine B isothiocyanate     

Drosophila protein phosphatase 5 is encoded by a single gene that is most highly expressed during embryonic development

A putative Drosophila melanogaster homologue of mammalian PP5, termed Dm PP5, was identified from cDNA. Dm PP5 comprises a phosphatase catalytic domain preceded by an amino terminal domain containing three tetratricopeptide repeat motifs and shares 60% overall amino acid identity with human PP5. Genomic restriction analysis identified a single Dm PP5 gene that was mapped to the third chromosome at locus 85E10-12 and a strain carrying a deletion that encompasses this gene was identified. Dm PP5 mRNA and protein are more highly expressed in the embryo than at later developmental stages, but their expression levels do not always change synchronously. Dm PP5 protein localises to both the nucleus and the cytoplasm of cells at the periphery of newly cellularized embryos.     

An endoplasmic reticulum protein, p180, is highly expressed in human cytomegalovirus-permissive cells and interacts with the tegument protein encoded by UL48

We have used a virus overlay assay to detect cellular proteins associated with human cytomegalovirus (HCMV) particles. The radiolabeled HCMV particles specifically bound to two host proteins with molecular sizes of 150 and 180 kDa. By a micro-amino-acid sequencing technique, the 180-kDa protein was identified as a human homologue of the ES130/p180 ribosome receptor (p180), which is an integral endoplasmic reticulum (ER) membrane protein possessing a very unique tandem repeat domain at its N-terminal region. The virus overlay assay using truncated p180 polypeptides revealed that HCMV binding to human p180 occurred through the N-terminal region. In HCMV-permissive cells the high level of expression of the human p180 protein was clearly observed regardless of cell type. Furthermore, we showed that p180 binds to the UL48 gene product, which is one of the predominant tegument proteins of HCMV and which is considered to be tightly associated with the capsid. The interaction between the two proteins was assumed to be specific and was observed both in vitro and in vivo. During the late phase of infection, the unique relocation of human p180 was observed, that is, to the juxtanuclear region, which appeared to be in the vicinity of the area where naked virions were frequently observed in an electron-microscopic study. Thus our data suggest that p180 interacts with the HCMV tegument, at least through pUL48, during the HCMV replication process. We discuss the possible role of the interaction between p180 and pUL48 in the intracellular transport of HCMV virions.     

Ligand-dependent secretion of rat retinol-binding protein expressed in HeLa cells.

A minigene encoding rat retinol-binding protein (RBP) was transfected into HeLa cells, which do not express endogenous RBP, transthyretin, or cellular retinol-binding protein. The HeLa cells manufactured and secreted the transfected gene product, demonstrating that RBP-transthyretin assembly is not a requirement for the secretion of RBP. When HeLa cells were grown under vitamin A-deficient conditions, RBP accumulated in the endoplasmic reticulum. Both serum and retinol stimulated secretion of RBP in a concentration-dependent manner. The retinol-regulated secretion occurred also after protein synthesis had been blocked by cycloheximide. Addition of holo-RBP or retinal, but not retinoic acid, stimulated secretion of RBP. Thus, an in vitro model system that resembles the rat hepatocyte in vivo with regard to the known regulation of RBP secretion has been established in a human cell line of extrahepatic origin. It can be concluded that cellular retinol-binding protein is not required for the transfer of retinol to RBP and that the mechanism whereby retinol controls the intracellular transport of RBP is neither specific for tissues synthesizing RBP nor species-specific. To investigate the structural properties responsible for the endoplasmic reticulum retention of RBP in the absence of its ligand, a cDNA encoding chicken purpurin, a protein that is 50% identical to RBP and that binds retinol, was expressed in HeLa cells. In contrast to RBP, purpurin was not retained in vitamin A-deficient HeLa cells.     

Biochemical characterization of a new recombinant TNF receptor-hyFc fusion protein expressed in CHO cells

The currently used Tumor Nectosis Factor (TNF)-α blockers such as infliximab, adalimumab and etanercept have Fc regions of the human IgG1 subtype have advantages in terms of in vivo half-life, however these could raise potential concerns for unwanted effector-mediated effects, such as antibody dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). To address this issue, we constructed a novel hybrid protein with decreased ADCC and CDC potentials by fusing the TNF receptor to a hybrid Fc (hyFc) containing CH2 and CH3 regions of IgG4 and highly flexible hinge regions of IgD which neither has ADCC and CDC activities. The resulting fusion protein, TNFR-hyFc, was over-expressed in CHO cells. For use as a pre-clinical material in pharmacology, PK and toxicological evaluations were carried out for biochemical characterization which was then compared with etanercept that has similarity in structure. Amino acid composition analysis and peptide mapping showed that the expressed TNFR-hyFc matched the theoretical composition derived from the DNA sequence. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) showed that TNFR-hyFc is 2.9 kDa larger than etanercept. MALDI-TOF after removal of N-glycans by PNGase treatment showed that TNFR-hyFc is 3.9 kDa larger than etanercept. Isoelectric focusing and monosaccharide analysis showed that TNFR-hyFc is slightly more acidic than etanercept. N-terminal amino acid sequencing showed that N-terminal heterogeneity is present in both TNFR-hyFc and etanercept, although the ratios are somewhat different. Glycan analysis showed that the main glycan form is bi-antennary, similar to etanercept.     

Extracellular accumulation of recombinant protein by Escherichia coli in a defined medium

Extracellular accumulation of recombinant proteins in the culture medium of Escherichia coli is desirable but difficult to obtain. The inner or cytoplasmic membrane and the outer membrane of E. coli are two barriers for releasing recombinant proteins expressed in the cytoplasm into the culture medium. Even if recombinant proteins have been exported into the periplasm, a space between the outer membrane and the inner membrane, the outer membrane remains the last barrier for their extracellular release. However, when E. coli was cultured in a particular defined medium, recombinant proteins exported into the periplasm could diffuse into the culture medium automatically. If a nonionic detergent, Triton X-100, was added in the medium, recombinant proteins expressed in the cytoplasm could also be released into the culture medium. It was then that extracellular accumulation of recombinant proteins could be obtained by exporting them into the periplasm or releasing them from the cytoplasm with Triton X-100 addition. The tactics described herein provided simple and valuable methods for achieving extracellular production of recombinant proteins in E. coli.