Novel cell line development strategy for monoclonal antibody manufacturing using translational enhancing technology.

Chinese hamster ovary (CHO) cells are widely used for constructing expression systems to produce therapeutic proteins. However, the establishment of high-producer clones remains a laborious and time-consuming process, despite various progresses having been made in cell line development. We previously developed a new strategy for screening high monoclonal antibody (mAb)-producing cells using flow cytometry (FCM). We also reported that p180 and SF3b4 play key roles in active translation on the endoplasmic reticulum, and that the productivity of secreted alkaline phosphatase was enhanced by the overexpression of p180 and SF3b4. Here, we attempted to apply the translational enhancing technology to high mAb-producing cells obtained after high-producer cell sorting. A high mAb-producing CHO clone, L003, which showed an mAb production level of >3 g/L in fed-batch culture, was established from a high mAb-producing cell pool fractionated by FCM. Clones generated by the overexpression of p180 and SF3b4 in L003 cells were evaluated by fed-batch culture. The specific productivity of clones overexpressing these two factors was ∼3.1-fold higher than that of parental L003 cells in the early phase of the culture period. Furthermore, the final mAb concentration was increased to 9.5 g/L during 17 days of fed-batch culture after optimizing the medium and culture process. These results indicate that the overexpression of p180 and SF3b4 would be promising for establishing high-producer cell lines applicable to industrial production.

Oil spill remediation by using the remediation agent JE1058BS that contains a biosurfactant produced by Gordonia sp. strain JE-1058.

A remediation agent containing a biosurfactant was prepared by spray drying the sterilized culture broth of Gordonia sp. strain JE-1058, and the agent was designated as JE1058BS. On subjection to the baffled flask test developed by the United States Environmental Protection Agency, JE1058BS showed a strong potential to be applied as an oil spill dispersant even in the absence of a solvent. It also proved to be an effective bioremediation agent for the remediation of oil spills at sea. The addition of JE1058BS to seawater stimulated the degradation of weathered crude oil (ANS 521) via the activity of the indigenous marine bacteria. Its addition also stimulated the removal of crude oil from the surface of contaminated sea sand. These results indicate that biosurfactant-containing JE1058BS has a strong potential to be applied as a remediation agent for the clean-up of oil spills at sea and on shorelines.

Analysis of the 7.6-kb cryptic plasmid pNC500 from Rhodococcus rhodochrous B-276 and construction of Rhodococcus-E. coli shuttle vector.

Four circular cryptic plasmids were detected from propene-degrading Rhodococcus rhodochrous (formerly Nocardia corallina) B-276 and the smallest 7.6-kb plasmid, named pNC500 was used to construct Rhodococcus-E. coli shuttle vector, pNC5403. Sequence analysis of pNC500 revealed that the plasmid contains eight potential ORFs, namely 1 through 8. The deduced amino acid sequences for ORFs 3, 4, 6, and 7 show homology with those of Rep A, Rep B, DNA methyl-transferase (M.XamI), and restriction nuclease (R.XamI), respectively. The region responsible for replication in the potent oil-desulfurization bacterium, Rhodococcus opacus T09 was determined as 3.7 kb-XbaI/BalI fragment which contains ORFs 3 and 4, while no transformants were obtained when ORF 4 was partially deleted, suggesting that both are required for its replication. Alignment of the predicted amino acid sequences revealed that ORFs 3 and 4 were DNA binding protein and DNA primase, respectively. A compatibility test with pAL5000-related plasmid vector, pRHK1, which contains pRC4, revealed that pNC5403 was compatible with pRHK1 suggesting that each replication origin would be different. ORFs 3 and 4 containing a pNC5403 derivative, pN5DXB, was stably maintained for over 80 generations in the absence of antibiotic selective conditions.

Characterization of Rhodococcus-E. coli shuttle vector pNC9501 constructed from the cryptic plasmid of a propene-degrading bacterium.

Rhodococcus-E. coli shuttle vector pNC9501 was constructed using circular cryptic plasmid pNC903 from propene-degrading Rhodococcus ruber P-II-123-1. Sequence analysis of pNC903 revealed two open-reading frames encoding the replication proteins Reps A and B. In the amino acid sequence of the putative Rep B, a helix-turn-helix motif, which is responsible for the binding of DNA, was found. Sequencing of the upstream region of the putative Rep A and incompatibility tests revealed that pNC903 is a Mycobacterium-derived pAL5000-related plasmid. pNC9501 could also be transformed into Mycobacterium sp. showing good segregation stability (<0.1% plasmid loss/generation) in the absence of selective pressure.

Degradation of trichloroethene by a linear-plasmid-encoded alkene monooxygenase in Rhodococcus corallinus (Nocardia corallina) B-276.

Rhodococcus corallinus (formerly Nocardia corallina) B-276, isolated with propene as sole carbon and energy source, is able to oxidize trichloroethene (TCE). Glucose- or propene-grown R. corallinus B-276 cells exhibited no difference in TCE degradation efficiency. TCE degradation was found to be growth-phase-dependent and maximum rates were monitored with stationary-phase cells. K(m) and Vmax values for TCE degradation of R. corallinus B-276 grown in nutrient broth medium in the presence of glucose were 187 microM and 2.4 nmol min-1 (mg protein)-1, respectively. Escherichia coli recombinants harbouring and expressing the alkene monooxygenase genes of R. corallinus B-276 exhibited the ability to degrade TCE. This result provides clear evidence that the alkene monooxygenase of R. corallinus B-276 catalyses TCE oxidation. R. corallinus B-276 was shown to contain four linear plasmids, pNC10 (70 kb), pNC20 (85 kb), pNC30 (185 kb) and pNC40 (235 kb). The observation that pNC30-deficient strains had lost the ability to grow on propene suggested that the genes of the propene degradation pathway are encoded by the linear plasmid pNC30. Southern blot analysis with cloned alkene monooxygenase genes from R. corallinus B-276 revealed a positive hybridization signal with the linear plasmid pNC30. This result clearly shows that the alkene monooxygenase is encoded by the linear plasmid pNC30. Eleven short-chain-alkene-oxidizing strains were screened for the presence of linear plasmids. Among these, four propene-oxidizing Rhodococcus strains and one ethene-oxidizing Mycobacterium strain were found to contain linear megaplasmids. Southern blot analysis with the alkene monooxygenase revealed positive signals with linear plasmids of two propene-oxidizing Rhodococcus ruber strains. These results indicate that homologous alkene monooxygenases are encoded by linear plasmids in R. ruber strains.