Takeshi Omasa

Effect of methanol concentration on the production of human β2-glycoprotein I domain V by a recombinant Pichia pastoris: A simple system for the control of methanol concentration using a semiconductor gas sensor

Abstract The methylotrophic yeast Pichia pastoris is one of the best hosts for the production of foreign proteins because of the presence of the strong AOX1 promoter induced by methanol. Methanol feeding during the production phase of the foreign proteins is important because methanol not only induces protein production but also provides energy source for the host cells. Excess methanol inhibits the growth of host cells, while an insufficient amount of energy source and/or methanol starvation lead to poor growth and production. We constructed a simple methanol control system consisting of a semiconductor gas sensor and a relay. Using this system, we studied the effect of methanol concentration on the production of a model foreign protein, human β2-glycoprotein I domain V. The methanol concentrations were kept constant at 1.5, 10, 17, or 31 g·l−1 (±5%) during the production phase. Although the specific rates of growth and methanol consumption decreased with increase in the methanol concentration, the specific production rates increased, indicating that the energy for the production competed with that for cell growth. Accordingly, we provided glycerol as an extra energy source during the production phase, with the result that the specific production rate increased two times. Our simple and inexpensive system will help bioengineering studies on the production of recombinant proteins in P. pastoris, the growth and production of objective proteins in which are dependent on the methanol concentration.

Cell engineering and cultivation of chinese hamster ovary (CHO) cells.

Mammalian cell lines are important host cells for the industrial production of pharmaceutical proteins owing to their capacity for correct folding, assembly and post-translational modification. In particular, Chinese hamster ovary (CHO) cells are the most dependable host cells for the industrial production of therapeutic proteins. Growing demand for therapeutic proteins promotes the development of technologies for high quality and productivity in CHO expression systems. The following are fundamentally important for effective production. 1) Construction of cultivation process. The CHO-based cultivation process is well established and is a general platform of therapeutic antibody production. The cost of therapeutic protein production using CHO cells is equivalent to that using microbial culture. 2) Cell line development. Recent developments in omics technologies have been essential for the development of rational methods of constructing a cell line. 3) Cell engineering for post-translational steps. Improvement of secretion, folding and glycosylaiton is an important key issue for mammalian cell production systems. This review provides an overview of the industrial production of therapeutic proteins using a CHO cell expression system.

Amplified Gene Location in Chromosomal DNA Affected Recombinant Protein Production and Stability of Amplified Genes

Previously, we established an easy and quick construction method for obtaining a stable and highly productive gene‐amplified recombinant Chinese hamster ovary (CHO) cell line. With a gradual increase in methotrexate (MTX) concentration, gene‐amplified cell pools had high and stable specific growth and production rates. Moreover, the phenotype of gene‐amplified cells seemed to be affected by the location of the amplified gene in chromosomal DNA. We suspected that various kinds of gene‐amplified cells might appear during the long‐term selection to construct gene‐amplified cell pools. To clarify the behavior of gene‐amplified cell pools during a stepwise increase of MTX concentration, we isolated gene‐amplified clones derived from gene‐amplified cell pools. We compared the characteristics of isolated clones, such as the productivity of recombinant protein, stability of amplified genes, and the location of amplified genes. As a result, telomere‐type clones, in which the amplified gene was located near the telomeric region, were found to be more stable and productive than other types of clones. Telomere‐type clones had over 100 copies of amplified genes in the chromosomal DNA. In contrast, a large number of other types of clones had less than 10 copies of amplified genes. During long‐term cultivation in the absence of MTX, in other types of clones, amplified genes rapidly decreased in the chromosomal DNA.

Synthetic metabolic engineering-a novel, simple technology for designing a chimeric metabolic pathway.

BackgroundThe integration of biotechnology into chemical manufacturing has been recognized as a key technology to build a sustainable society. However, the practical applications of biocatalytic chemical conversions are often restricted due to their complexities involving the unpredictability of product yield and the troublesome controls in fermentation processes. One of the possible strategies to overcome these limitations is to eliminate the use of living microorganisms and to use only enzymes involved in the metabolic pathway. Use of recombinant mesophiles producing thermophilic enzymes at high temperature results in denaturation of indigenous proteins and elimination of undesired side reactions; consequently, highly selective and stable biocatalytic modules can be readily prepared. By rationally combining those modules together, artificial synthetic pathways specialized for chemical manufacturing could be designed and constructed.ResultsA chimeric Embden-Meyerhof (EM) pathway with balanced consumption and regeneration of ATP and ADP was constructed by using nine recombinant E. coli strains overproducing either one of the seven glycolytic enzymes of Thermus thermophilus, the cofactor-independent phosphoglycerate mutase of Pyrococcus horikoshii, or the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase of Thermococcus kodakarensis. By coupling this pathway with the Thermus malate/lactate dehydrogenase, a stoichiometric amount of lactate was produced from glucose with an overall ATP turnover number of 31.ConclusionsIn this study, a novel and simple technology for flexible design of a bespoke metabolic pathway was developed. The concept has been testified via a non-ATP-forming chimeric EM pathway. We designated this technology as “synthetic metabolic engineering”. Our technology is, in principle, applicable to all thermophilic enzymes as long as they can be functionally expressed in the host, and thus would be potentially applicable to the biocatalytic manufacture of any chemicals or materials on demand.

Gene amplification and its application in cell and tissue engineering

Gene amplification means the repeated replication of a certain gene without a proportional increase in the copy number of other genes and is a widespread phenomenon in eukaryotes. It is an important developmental and evolutionary process in many organisms. This article focuses on mammalian gene amplification and its application in cell and tissue engineering. The dhfr gene amplification in Chinese hamster ovary (CHO) cells, the gene amplification mechanism, the selection protocol and the application of gene amplification were reviewed.

Construction of BAC‐based physical map and analysis of chromosome rearrangement in chinese hamster ovary cell lines

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology for many years as a mammalian host cell platform for cloning and expressing genes of interest. A detailed physical chromosomal map of the CHO DG44 cell line was constructed by fluorescence in situ hybridization (FISH) imaging using randomly selected 303 BAC clones as hybridization probes (BAC‐FISH). The two longest chromosomes were completely paired chromosomes; other chromosomes were partly deleted or rearranged. The end sequences of 624 BAC clones, including 287 mapped BAC clones, were analyzed and 1,119 informative BAC end sequences were obtained. Among 303 mapped BAC clones, 185 clones were used for BAC‐FISH analysis of CHO K1 chromosomes and 94 clones for primary Chinese hamster lung cells. Based on this constructed physical map and end sequences, the chromosome rearrangements between CHO DG44, CHO K1, and primary Chinese hamster cells were investigated. Among 20 CHO chromosomes, eight were conserved without large rearrangement in CHO DG44, CHO K1, and primary Chinese hamster cells. This result suggested that these chromosomes were stable and essential in CHO cells and supposedly conserved in other CHO cell lines. Biotechnol. Bioeng. 2012; 109:1357–1367.

Evaluation of stable and highly productive gene amplified CHO cell line based on the location of amplified genes

In order to establish an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, variouskinds of stepwise methotrexate (MTX) selection were carriedout. The specific growth and production rates of the cell were compared with each other, and the distribution of the amplified gene location was determined using fluorescence in situ hybridization (FISH). The specific growth andproduction rates of the cell pool reached the highest levels under the selection condition in which the stepwise increase in the MTX concentration was most gradual; about 82% of amplified genes were observed near the telomeric region. During long-term cultivation without MTX, the percentage ofamplified genes near the telomeric region hardly changed, butthat of amplified genes at other regions decreased. Based on these results, stable and highly productive cell pools could be easily and quickly constructed and amplified and gradual stepwise increase of the MTX concentration. In addition, the FISH technique was powerful tool to evaluate highly productiveand stable gene-amplified cells based on the chromosomal location of the amplified gene.

The enhancement of specific antibody production rate in glucose- and glutamine-controlled fed-batch culture.

The concentration effects of certain amino acids (Asp, Ile, Leu, Lys, Met, Val, Phe and Gln which were highly consumed during cultivation), and glucose on cell growth and antibody productivity were investigated using dish culture. From these experiments, it was found that only glutamine enrichment enhanced the specific antibody production rate. The other amino acids described above did not affect either the specific growth rate or specific antibody production rate. Thus we investigated the quantitative effects of glutamine concentration in the range of 0.4∼33.3 mmol·1−1 on kinetic parameters in fed-batch culture which kept both glucose and glutamine concentration constant. As a result the specific growth rate decreased with increase in glutamine concentration in the range larger than 20 mmol·1−1. The specific antibody production rate had a maximum value at about 25 mmol·1−1 glutamine concentration.

Overexpression of GADD34 Enhances Production of Recombinant Human Antithrombin III in Chinese Hamster Ovary Cells

To improve the production of recombinant human antithrombin III (AT-III) in Chinese hamster ovary (CHO) cells, the gene encoding growth arrest and DNA damage inducible protein 34 (GADD34), which is a transcription factor involved in the unfolded protein response (UPR), was cloned from CHO-K1 cells. Overexpression of GADD34 significantly enhanced the production of recombinant AT-III in CHO 13D-35D cells. The specific rate of AT-III production in the GADD34-overexpressing CHO 13D-35D cells reached approximately 28 pg/cell/d. After 144 h of incubation, the AT-III concentration in the culture supernatant was approximately 40% higher than that observed in the case of the parental CHO 13D-35D cells. The mRNA expression, specific activity, and fucosylation of AT-III were not affected by GADD34 overexpression. Overexpression of GADD34 is a promising method of improving the production of secreted protein pharmaceuticals in CHO cells.

In vivo estimation of bioartificial liver with recombinant HepG2 cells using pigs with ischemic liver failure.

Biological efficacy of a recombinant human hepatic cell line, glutamine synthetase transfected HepG2 (GS-HepG2), was examined with large-scale culture in a circulatory flow bioreactor and in pigs with ischemic liver failure. GS-HepG2 cells were cultured in a circulatory flow bioreactor from 5 × 107 to 4 × 109 cells for 109 days. The cells showed ammonia removal activity even under substrate (glutamic acid)-free medium, suggesting that the GS catalyzed the activity using intracellular glutamic acid that had been pooled during conventional culture. When GS-HepG2 bioartificial liver (BAL) was applied to pigs with ischemic liver failure, survival time was prolonged to 18.8 ± 6.1 h (mean ± SD, n = 4) from 13.8 ± 5.4 h (n = 6) and 10.7 ± 4.1 h (n = 6) (groups treated with cell-free BAL and treated with plasma exchange and continuous hemodia-filtration, respectively). Laboratory data indicated the tendency for improvement in increase of blood ammonia level and decline of blood coagulation indices in the GS-HepG2 BAL-treated group. The advantages and potential for the cell line as a bioreactor in BAL is also discussed, comparing to those of isolated porcine hepatocytes.