R-protein Production Research Articles

Long term culture promotes changes to growth, gene expression, and metabolism in CHO cells that are independent of production stability.

Phenotypic stability of Chinese hamster ovary (CHO) cells over long term culture (LTC) presents one of the most pressing challenges in the development of therapeutic protein manufacturing processess. However, our current understanding of the consequences of LTC on recombinant (r-) CHO cell lines is still limited, particularly as clonally-derived cell lines present distinct production stability phenotypes. This study evaluated changes of culture performance, global gene expression, and cell metabolism of two clonally-derived CHO cell lines with a stable or unstable phenotype during the LTC (early [EP] vs. late [LP] culture passages). Our findings indicated that LTC altered the behavior of CHO cells in culture, in terms of growth, overall gene expression, and cell metabolism. Regardless whether cells were categorized as stable or unstable in terms of r-protein production, CHO cells at LP presented an earlier decline in cell viability and loss of any observable stationary phase. These changes were parallelled by the upregulation of genes involved in cell proliferation and survival pathways (i.e., MAPK/ERK, PI3K-Akt). Stable and unstable CHO cell lines both showed increased consumption of glucose and amino acids at LP, with a parallel accumulation of greater amounts of lactate and TCA cycle intermediates. In terms of production stability, we found that decreased r-protein production in the unstable cell line directly correlated to the loss in r-gene copy number and r-mRNA expression. Our data revealed that LTC produced ubiquitious effects on CHO cell phenotypes, changes that were rooted in alterations in cell transcriptome and metabolome. Overall, we found that CHO cells adapted their cellular function to proliferation and survival during the LTC, some of these changes may well have limited effects on overall yield or specific productivity of the desired r-product, but they may be critical towardthe capacity of cells to handle r-proteins with specific molecular features.

Combined gene and environmental engineering offers a synergetic strategy to enhance r‐protein production in Chinese hamster ovary cells

Environmental growth-inhibition conditions (GICs) have been used extensively for increasing cell-specific productivity (qP ) of Chinese hamster ovary (CHO) cells, with the most common being temperature downshift and sodium butyrate (NaBu) treatment. B lymphocyte-induced maturation protein-1 (BLIMP1) overexpression in CHO cells can also inhibit cell growth and increase product titers and qP . Given the similar responses, this study evaluated the individual and combined effects of BLIMP1 expression, low temperature, and NaBu treatment on culture performance, cell metabolism, and recombinant protein production of CHO cells. As expected, all three interventions decreased cell growth, arrested cells in G1/G0 cell cycle phase, and increased qP . However, CHO cells presented different responses when considering cell viability, recombinant gene expression, and cell metabolism that indicated differences in the molecular loci by which BLIMP1 and GICs generated higher productivities. Combinations of BLIMP1 expression and GICs acted synergistically to inhibit cell growth and maximize r-protein production, with the BLIMP1/NaBu condition leading to the most significant improvements in product titers and qP . This latter condition also proved to substantially increase product yields (up to 9.8 g immunoglobulin G1 [IgG1]/L and 2.2 g erythropoietin-Fc [EPO-Fc]/L) and qP (up to 179 pg/cell/day [pcd] for IgG1 and 30 pcd for EPO-Fc) in high-density perfusion cultures. These findings offered mechanistic insights about the productivity-enhancing effects of BLIMP1 and GICs, as well as their complementarity for generating highly productive processes.

Hybrid-architectured double-promoter expression systems enhance and upregulate-deregulated gene expressions in Pichia pastoris in methanol-free media.

Double-promoter expression system (DPES) design as de novo metabolic engineering strategy enables fine-tuned and enhanced gene expression. We constructed a collection of monodirectional hybrid-architectured DPESs with engineered promoter variants PADH2-Cat8-L2 and PmAOX1 and with the naturally occurring promoter PGAP to enhance and upregulate-deregulated gene expressions in Pichia pastoris in methanol-free media. Reporter red fluorescent protein (mApple) and enhanced green fluorescent protein (eGFP) were expressed under PADH2-Cat8-L2 and PmAOX1 or PGAP, respectively, enabling the determination of the transcription period and strength of each constituent in the DPESs. We determined fluorescent protein expressions in batch cultivations on 2% (v/v) ethanol, excess glucose, and excess glycerol, and compared them with single-promoter expression systems constructed with PADH2-Cat8-L2, PmAOX1, and PGAP. The transcription- and expression-upregulation power of bifunctional DPESs was higher than that of twin DPESs (two-copy expression systems). Our findings answer long-standing questions regarding the high- (or multi-) copy clone results in the literature. Our first conclusion is that increasing identical components in the DPES architectures linearly increases the concentrations of cis-acting DNA sites and increases the demand for key transcription factors (TFs) that perturb their good coupling of supply and demand. The next is that the synthesis of some amino acids may create bottleneck(s) as rate-limiting amino acid(s) in recombinant protein synthesis. With bifunctional DPESs, each constituent upregulated the transcription and increased the expression and reduced the demand for the same TF(s) in the generation of novel regulatory circuits, due to the increased number of nonidentical cis-acting DNA sites. We tested superior DPES performances in extracellular human growth hormone (rhGH) production. Thereby, the indications related to the rate-limiting amino acids were verified. Compared with its constituents PADH2-Cat8-L2 and PmAOX1, the bifunctional DPES4 enhanced rhGH production by 1.44- and 2.02-fold, respectively. The DPES design method, with its constraint and parameters, enables the generation of promising r-protein production platforms with high impact on industrial-scale production processes and opens up new avenues for research in yeasts. KEY POINTS: • Design method with the constraint and parameters for the construction of the DPESs is presented. • Hybrid-architectured de novo DPESs are designed to enhance and fine-tune gene expression. • Bifunctional DPESs demonstrate enhanced transcription and expression. • Twin DPESs linearly increase cis-acting DNA sites and consequently increase the demand for the same TFs. • Bifunctional DPESs enable good coupling of supply and demand to bind with TFs. • Ethanol-controlled Snf1 pathway and crosstalk enable fine-tuned transcription and enhanced expression.

Temperature Down-Shift Modifies Expression of UPR-/ERAD-Related Genes and Enhances Production of a Chimeric Fusion Protein in CHO Cells.

Low culture temperature enhances the cell-specific productivity of Chinese hamster ovary (CHO) cells expressing varied recombinant (r-) proteins, but the mechanisms remain unclear. Regulation of unfolded protein response (UPR) pathway genes, such as transcriptional regulatory factors and endoplasmic reticulum (ER)-resident proteins, appear to be involved in the improvements of r-protein production under low temperature conditions. The transcriptional regulation of UPR-specific targets is studied in response to decreased culture temperature in relation to production of a difficult-to-express protein. A clonally-derived CHO cell line expressing a chimeric fusion protein (human erythropoietin [hEPO] linked to a murine Fc region, hEPO-Fc) is evaluated in terms of growth, metabolism, r-protein production and UPR-/ER associated degradation (ERAD)-specific gene expression at standard (37°C) and low (32°C) temperature in batch and fed-batch systems. Low temperature decreased peak cell density, improved viability, generated cell cycle arrest in the G1 phase and enhanced hEPO-Fc expression in both batch and fed-batch cultures. A low culture temperature significantly upregulated genes encoding UPR-specific transcriptional activators (xbp1s, ddit3, and atf5) and ER-resident proteins (grp78, grp94, trib3, and ero1α), that are associated with folding and processing of proteins within the ER. Further, low culture temperature decreased expression of genes involved in ERAD (edem3, sels, herpud1, and syvn1) indicating a decreased potential for protein degradation.

Metabolic flux analysis during galactose and lactate co-consumption reveals enhanced energy metabolism in continuous CHO cell cultures

Chinese Hamster Ovary (CHO) cells are the main expression system for production of therapeutic recombinant (r-) proteins. The increased demand for these therapeutics has boosted the development of more robust production processes. Use of optimised feed/media has dramatically improved the performance of CHO cells in culture. However, such progress in biomass synthesis and r-protein production often come with an increased accumulation of lactate. In this study, we present a combined feeding strategy that uses galactose and lactate to replace glucose in CHO cell cultures. Replacement of glucose by galactose and lactate sustained cell growth and r-protein production in CHO cells. This strategy supported a better-balanced and more efficient metabolism, observed by an overall decreased consumption of carbon sources and amino acids, associated with an increased ATP production per C-mol consumed. Our results provide new insights of CHO cell metabolism in glucose-free media based on galactose and lactate.

High glucose and low specific cell growth but not mild hypothermia improve specific r-protein productivity in chemostat culture of CHO cells.

In the biopharmaceutical sector, Chinese hamster ovary (CHO) cells have become the host of choice to produce recombinant proteins (r-proteins) due to their capacity for correct protein folding, assembly, and posttranslational modification. However, the production of therapeutic r-proteins in CHO cells is expensive and presents insufficient production yields for certain proteins. Effective culture strategies to increase productivity (qp) include a high glucose concentration in the medium and mild hypothermia (28–34 °C), but these changes lead to a reduced specific growth rate. To study the individual and combined impacts of glucose concentration, specific growth rate and mild hypothermia on culture performance and cell metabolism, we analyzed chemostat cultures of recombinant human tissue plasminogen activator (rh-tPA)-producing CHO cell lines fed with three glucose concentrations in feeding media (20, 30 and 40 mM), at two dilution rates (0.01 and 0.018 1/h) and two temperatures (33 and 37 °C). The results indicated significant changes in cell growth, cell cycle distribution, metabolism, and rh-tPA productivity in response to the varying environmental culture conditions. High glucose feed led to constrained cell growth, increased specific rh-tPA productivity and a higher number of cells in the G2/M phase. Low specific growth rate and temperature (33 °C) reduced glucose consumption and lactate production rates. Our findings indicated that a reduced specific growth rate coupled with high feed glucose significantly improves r-protein productivity in CHO cells. We also observed that low temperature significantly reduced qp, but not cell growth when dilution rate was manipulated, regardless of the glucose concentration or dilution rate. In contrast, we determined that feed glucose concentration and consumption rate were the dominant aspects of the growth and productivity in CHO cells by using multivariate analysis.

Double promoter expression systems for recombinant protein production by industrial microorganisms.

Using double promoter expression systems is a promising approach to increase heterologous protein production. In this review, current double promoter expression systems for the production of recombinant proteins (r-proteins) by industrially important bacteria, Bacillus subtilis and Escherichia coli; and yeasts, Saccharomyces cerevisiae and Pichia pastoris, are discussed by assessing their potentials and drawbacks. Double promoter expression systems need to be designed to maintain a higher specific product formation rate within the production domain. While bacterial double promoter systems have been constructed as chimeric tandem promoters, yeast dual promoter systems have been developed as separate expression cassettes. To increase production and productivity, the optimal transcriptional activity should be justified either by simultaneously satisfying the requirements of both promoters, or by consecutively stimulating the changeover from one to another in a biphasic process or via successive-iterations. Thus, considering the dynamics of a fermentation process, double promoters can be classified according to their operational mechanisms, as: i) consecutively operating double promoter systems, and ii) simultaneously operating double promoter systems. Among these metabolic design strategies, extending the expression period with two promoters activated under different conditions, or enhancing the transcriptional activity with two promoters activated under similar conditions within the production domain, can be applied independently from the host. Novel studies with new insights, which aim a rational systematic design and construction of dual promoter expression vectors with tailored transcriptional activity, will empower r-protein production with enhanced production and productivity. Finally, the current state-of-the-art review emphasizes the advantages of double promoter systems along with the necessity for discovering new promoters for the development of more effective and adaptive processes to meet the increasing demand of r-protein industry.

Fluctuations and synchrony of RNA synthesis in nucleoli.

Ribosomal RNA (rRNA) sequences are synthesized at exceptionally high rates and, together with ribosomal proteins (r-proteins), are utilized as building blocks for the assembly of pre-ribosomal particles. Although it is widely acknowledged that tight regulation and coordination of rRNA and r-protein production are fundamentally important for the maintenance of cellular homeostasis, still little is known about the real-time kinetics of the ribosome component synthesis in individual cells. In this communication we introduce a label-free MicroRaman spectrometric approach for monitoring rRNA synthesis in live cultured cells. Remarkably high and rapid fluctuations of rRNA production rates were revealed by this technique. Strikingly, the changes in the rRNA output were synchronous for ribosomal genes located in separate nucleoli of the same cell. Our findings call for the development of new concepts to elucidate the coordination of ribosomal components production. In this regard, numerical modeling further demonstrated that the production of rRNA and r-proteins can be coordinated, regardless of the fluctuations in rRNA synthesis. Overall, our quantitative data reveal a spectacular interplay of inherently stochastic rates of RNA synthesis and the coordination of gene expression.

A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1.

Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 μg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications.

In-silico determination of Pichia pastoris signal peptides for extracellular recombinant protein production

In-silico identified novel secretory signal peptides (SPs) are required in vivo to achieve efficient transfer or to prevent other cellular proteins from interfering with the process in extracellular recombinant protein (r-protein) production. 56 endogenous and exogenous secretory SPs, have been used or having the potential to be used in Pichia pastoris for r-protein secretion, were analyzed in-silico using the softwares namely SignalP4.1, Phobius, WolfPsort0.2, ProP1.0, and NetNGlyc1.0. Among the predicted 41 endogenous secretory SPs, five of them have been used in P. pastoris, and regarded as positive controls; whereas, 36 of them have not been used. Amongst, the predicted cleavage site for each of the 32 endogenous secretory SPs was found to be same by the three programs. The secretory SPs having the highest D-scores, the score quantifying the signal peptide-ness of a given sequence segment, were: MKILSALLLLFTLAFA (D=0.932), MRPVLSLLLLLASSVLA (D=0.932), MFKSLCMLIGSCLLSSVLA (D=0.918). As D-scores of these SPs are higher than that of Saccharomyces cerevisiae α-mating factor signal peptide which has been widely used for r-protein production, they can be considered as the promising candidates. Among the predicted 15 exogenous SPs, 11 have been used in P. pastoris: therefore, they were evaluated as positive controls. The three programs predicted a unique cleavage site for each of the 10 exogenous SPs; and D-scores of these SPs were within D=0.805–0.900; whereas, four exogenous secretory SPs, which have not been used in P. pastoris, have D-scores within D=0.494–0.702.

Reduction in Ribosomal Protein Synthesis Is Sufficient To Explain Major Effects on Ribosome Production after Short-Term TOR Inactivation in Saccharomyces cerevisiae

Ribosome synthesis depends on nutrient availability, sensed by the target of rapamycin (TOR) signaling pathway in eukaryotes. TOR inactivation affects ribosome biogenesis at the level of rRNA gene transcription, expression of ribosomal proteins (r-proteins) and biogenesis factors, preribosome processing, and transport. Here, we demonstrate that upon TOR inactivation, levels of newly synthesized ribosomal subunits drop drastically before the integrity of the RNA polymerase I apparatus is severely impaired but in good correlation with a sharp decrease in r-protein production. Inhibition of translation by cycloheximide mimics the rRNA maturation defect observed immediately after TOR inactivation. Both cycloheximide addition and the depletion of individual r-proteins also reproduce TOR-dependent nucleolar entrapment of specific ribosomal precursor complexes. We suggest that shortage of newly synthesized r-proteins after short-term TOR inactivation is sufficient to explain most of the observed effects on ribosome production.

Utilizing Protein-Lean Coproducts from Corn Containing Recombinant Pharmaceutical Proteins for Ethanol Production

Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127-139 and 138-155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.

A structured kinetic model for recombinant protein production by Mut+ strain of Pichia pastoris

A structured kinetic biochemical model for Pichia pastoris growth and recombinant protein production, able to predict r-protein production and optimize the feeding strategy is reported. Pichia pastoris was divided into three compartments, the recombinant product human erythropoietin (rHuEPO) was considered in one compartment, alcohol oxidase and extracellular protease enzymes were assembled in the enzymatic compartment, and rest of the cell components (including intracellular proteases) were gathered in the cellular compartment. After parameter identification by the simplex method, sensitivity analysis was carried out to confirm that the global optimum values were reached for the parameters of the model. The model successfully simulated the dynamic changes and the resulting rHuEPO production caused by variations in methanol feeding rate, even in the lag and the stationary phases. The model successfully predicted the optimal feeding strategies and has great potential for use in process simulation and control of glycosylated recombinant protein production by Pichia pastoris cells.

Translational enhancement of recombinant protein synthesis in transgenic silkworms by a 5′-untranslated region of polyhedrin gene of Bombyx mori Nucleopolyhedrovirus

Previously, we established a method to produce recombinant proteins (r-proteins) in cocoons of germline transgenic silkworms, and showed that a step(s) in post-transcription processes was rate-limiting in obtaining a high yield of r-proteins. In this study, we examined whether the 5'-untranslated region (5'-UTR) of the polyhedrin gene (pol) of nucleopolyhedrovirus (NPV) has a translational enhancer activity in the r-protein expression by middle silk gland (MSG) cells of silkworm Bombyx mori (Bm). Sericin 1 gene (ser1) promoter-driven transformation vectors were constructed in which pol5'-UTRs of NPVs isolated from four different species, Bm, Spodoptera frugiperda, Ectropis oblique, and Malacosoma neustria, were each placed upstream of a reporter gene. Transient expression assays in MSGs showed that these pol5'-UTRs all enhanced the protein expression of reporter genes, and the pol5'-UTR of Bm NPV (pol5'-UTR/Bm) was the most effective among them. Thus, transgenic silkworms were generated, which bore the ser1 promoter-driven His-tagged secretory EGFP (sEGFP-His) gene under the control of pol5'-UTR/Bm. The synthesis of sEGFP-His proteins in MSGs of the transgenic worms was approximately 1.5-fold higher than that in those bearing null vectors. However, its mRNA expression levels were 67% of the control worms, indicating that the pol5'-UTR/Bm specifically enhanced the translational level. In conclusion, pol5'-UTR/Bm increased the yield of r-protein production in transgenic silkworms by enhancing the translational activity and this 5'-UTR could be useful for the mass production of r-proteins in germline transgenic silkworms.

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.

Monitoring of the Adenovirus Production Process by Flow Cytometry

Adenovirus (Ad) has become the vector of choice for gene therapy clinical protocols worldwide; it is the only viral vector to date that has been licensed for use in a gene therapy treatment. There is, however, a need to develop a simple, reliable at-line method to monitor the production of virus and recombinant proteins (r-proteins) that have no intrinsic reporter properties. Here we utilize flow cytometry to measure cell size, granularity, and DNA content in a single-step analysis and to correlate these parameters to the production of a type-5 Ad (Ad5) expressing the recombinant green fluorescent protein (GFP). Clear correlations between these parameters and productivity are made, with forward scatter and DNA content showing the highest correlation coefficients, 0.9 and 0.83 for virus production and r-protein production, respectively. Measuring these parameters requires little or no processing of the cells from culture to analysis. These parameters have been used successfully to monitor, at-line, the amount of Ad and r-protein product in a 293-Ad system.

Transient gene expression in HEK293 cells: Peptone addition posttransfection improves recombinant protein synthesis

Gene expression by large-scale transfection of mammalian cells is becoming an established technology for the fast production of milligram and even gram amounts of recombinant proteins (r-proteins). However, efforts are still needed to optimize production parameters in order to maximize volumetric productivities while maintaining product quality. In this study, transfection efficiency and volumetric productivity following transient gene expression in HEK293 cells were evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) as reporter genes. We show that a single pulse of peptones (protein hydrolysates) to the cultures performed in a low serum (1%, v/v) and in serum-free medium results in a significant increase in volumetric protein productivity. Sixteen peptones from different sources were tested and almost all of them showed a positive effect on r-protein production. This effect, however, is time- and concentration-dependent. By using Tryptone N1 (a casein peptone, TN1) to feed the cultures at 24 h posttransfection (hpt), a 2-fold increase in volumetric SEAP productivity was obtained 5 days posttransfection. This effect was shown to be equal to that obtained when the culture was fed with a supplementary 4% (v/v) of serum. The positive effect of TN1 on protein production was also demonstrated with Tie2 protein ectodomain produced in serum-free medium. HPLC analysis of amino acids consumption/production during control batch and TN1 pulse culture showed some major differences in amino acid metabolism when using TN1 pulse. Asparagine, glycine, histidine, threonine, leucine, and valine show accumulation in the medium over the cultivation period instead of being consumed as observed in unfed sample (except for asparagine, which remained unchanged). Isoleucine, tyrosine, methionine, and phenylalanine all remained unchanged or slightly fluctuated in TN1-fed culture after the feeding pulse, while they were all steadily consumed in the control run. The relative abundance of SEAP's mRNA suggests that the improvement in protein yield results both from an increase of the translational activity and transcription efficiency. Further understanding of mechanisms by which amino acids/peptides regulate transcriptional and translational machinery in mammalian cells should facilitate the design of new strategies for the improvement of r-protein production by large-scale transfection.

Effects of induction of rRNA overproduction on ribosomal protein synthesis and ribosome subunit assembly in Escherichia coli.

Overproduction of rRNA was artificially induced in Escherichia coli cells to test whether the synthesis of ribosomal protein (r-protein) is normally repressed by feedback regulation. When rRNA was overproduced more than twofold from a hybrid plasmid carrying the rrnB operon fused to the lambda pL promoter (pL-rrnB), synthesis of individual r-proteins increased by an average of about 60%. This demonstrates that the synthesis of r-proteins is repressed under normal conditions. The increase of r-protein production, however, for unknown reasons, was not as great as the increase in rRNA synthesis and resulted in an imbalance between the amounts of rRNA and r-protein synthesis. Therefore, only a small (less than 20%) increase in the synthesis of complete 30S and 50S ribosome subunits was detected, and a considerable fraction of the excess rRNA was degraded. Lack of complete cooperativity in the assembly of ribosome subunits in vivo is discussed as a possible explanation for the absence of a large stimulation of ribosome synthesis observed under these conditions. In addition to the induction of intact rRNA overproduction from the pL-rrnB operon, the effects of unbalanced overproduction of each of the two large rRNAs, 16S rRNA and 23S rRNA, on r-protein synthesis were examined using pL-rrnB derivatives carrying a large deletion in either the 23S rRNA gene or the 16S rRNA gene. Operon-specific derepression after 23S or 16S rRNA overproduction correlated with the overproduction of rRNA containing the target site for the operon-specific repressor r-protein. These results are discussed to explain the apparent coupling of the assembly of one ribosomal subunit with that of the other which was observed in earlier studies on conditionally lethal mutants with defects in ribosome assembly.

Transcriptional and translational regulation of ribosomal protein formation during mouse myoblast differentiation.

The metabolism of ribosomal proteins (r-proteins) and r-protein mRNAs was examined during mouse myoblast differentiation to identify the levels at which r-protein accumulation is regulated. Pulse-chase analyses of r-proteins in myoblasts and fibers indicate that the synthesis of r-proteins is coordinately reduced 2.0-fold following myoblast differentiation and that newly synthesized r-proteins do not turnover. This decreased synthesis of r-proteins in fibers is due to both a reduction in the steady-state levels of r-protein mRNAs and a decrease in the translational efficiency of r-protein mRNAs. Northern analyses of r-protein mRNA indicate that the steady-state levels of r-protein mRNAs S16, L18, and L32 are decreased 1.5-2.0-fold in fibers as compared to myoblasts. Analyses of the distribution of r-protein mRNAs in polysome gradients indicate that their translational efficiencies are reduced 1.3-1.6-fold in fibers as compared to myoblasts. To determine if the decrease in the steady-state levels of r-protein mRNAs is regulated at the level of transcription, the transcription of these genes was measured in isolated nuclei. These experiments show that the transcription of these r-protein genes is reduced 2-6-fold following myoblast differentiation. Thus, the production of r-proteins is regulated both at the level of transcription and translation during mouse myoblast differentiation.

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Previously, we established a method to produce recombinant proteins (r-proteins) in cocoons of germline transgenic silkworms, and showed that a step(s) in post-transcription processes was rate-limiting in obtaining a high yield of r-proteins. In this study, we examined whether the 5′-untranslated region (5′-UTR) of the polyhedrin gene (pol) of nucleopolyhedrovirus (NPV) has a translational enhancer activity in the r-protein expression by middle silk gland (MSG) cells of silkworm Bombyx mori (Bm). Sericin 1 gene (ser1) promoter-driven transformation vectors were constructed in which pol5′-UTRs of NPVs isolated from four different species, Bm, Spodoptera frugiperda, Ectropis oblique, and Malacosoma neustria, were each placed upstream of a reporter gene. Transient expression assays in MSGs showed that these pol5′-UTRs all enhanced the protein expression of reporter genes, and the pol5′-UTR of Bm NPV (pol5′-UTR/Bm) was the most effective among them. Thus, transgenic silkworms were generated, which bore the ser1 promoter-driven His-tagged secretory EGFP (sEGFP-His) gene under the control of pol5′-UTR/Bm. The synthesis of sEGFP-His proteins in MSGs of the transgenic worms was approximately 1.5-fold higher than that in those bearing null vectors. However, its mRNA expression levels were 67% of the control worms, indicating that the pol5′-UTR/Bm specifically enhanced the translational level. In conclusion, pol5′-UTR/Bm increased the yield of r-protein production in transgenic silkworms by enhancing the translational activity and this 5′-UTR could be useful for the mass production of r-proteins in germline transgenic silkworms.