Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors.

PURPOSE: The cyclin-dependent kinase 1 (Cdk1) and cyclin B complex performs important roles in the transition from the G2 to M phase in the cell cycle through removal of inhibitory phosphates on Cdk1, and Cdc25B, which is a dual-specific phosphatase, mediates these dephosphorylation events. However, measuring Cdc25B activity by existing methods is hampered by inadequate nonspecific substrates and the need to use a radiolabeled isotope. The present study aimed to develop an improved method with which to properly measure Cdc25B activity using a novel nonradioisotopic assay and Cdc25B overexpression cell lines. MATERIALS AND METHODS: A nonradioisotopic Cdk1 kinase assay, based on Western blotting for retinoblastoma protein and histone H1, was used to analyze Cdc25B activity. Also, stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines were constructed using the tetracycline-regulated expression system and were applied as a tool for screening for inhibitors of Cdc25B. RESULTS: The present study developed and optimized a nonradioisotopic assay method to properly measure Cdc25B activity. Furthermore, we constructed stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines for the establishment of a strong assay system with which to evaluate the specificity of Cdc25B inhibitors under conditions similar to the intracellular environment. These methods were confirmed as useful tools for measuring Cdc25B activity. CONCLUSION: The nonradioisotopic Cdk1 kinase assay and Cdc25B overexpression cell lines developed in this study can be conveniently used as tools for screening inhibitors of Cdc25B phosphatase as anticancer drugs.

Enhanced expression of soluble antibody fragments by low-temperature and overdosing with a nitrogen source.

Escherichia coli has been a primary host for the prokaryotic production of antibody fragments (Fabs) and has contributed to several successes in the pharmaceutical industry. Nevertheless, the requirement of disulfide bonds often results in low-yield fermentation and a lack of cost-effectiveness. Despite the improved production of functional Fabs by fermentation below 30 °C, the limited cellular growth needs further work. To address these issues, we investigated the effect of nitrogen supply on the cellular growth and the Fab productivity. We used the anti-human VEGF-A Fab as a model that exhibited poor expression at 37 °C regardless of the amount of nitrogen supplied during fermentation. In stark contrast, the expression yield of soluble Fab with a gross nitrogen supply of 6.91 g/L of broth throughout the fermentation at 25 °C was 332 mg/L. Furthermore, and increased nitrogen supply of 10.9 g/L significantly improved the yield of active form by 59.7% and the cellular growth rate by 39.3%. These results indicate that overdosing of a nitrogen source at low temperature is critical to Fab productivity in E. coli.

Purification of antibody fragments for the reduction of charge variants using cation exchange chromatography.

Recently, antibody fragments have been studied as therapeutic agents because they lack Fc effector function while having affinity similar to their original monoclonal antibody and can be produced using E. coli. Antibody fragments can be purified using affinity chromatography in the capture step, although they need a polishing step because of product-related impurities, mainly charge variants. Unlike monoclonal antibodies, few studies exist regarding the separation of charge variants in antibody variants. In this study, an efficient separation of charge variant method was assessed using a cation exchange chromatography resin with salt and a pH gradient. The SP ImpRes resin and pH gradient exhibited the most effective separation potency using combinations of resin and the separation method. The antibody fragment that did not undergo the charge variant separation process exhibited a difference in the tertiary structure of the protein and in vivo pharmacokinetics. However, the antibody fragment was similar to the reference protein when the charge variant separation process was performed. These results are expected to support efficient charge variant separation of antibody fragments and to be applied to the industrial production of therapeutic antibody fragments.

Cloning and characterization of the catabolite control protein A gene from Bacillus stearothermophilus No. 236.

The gene encoding the catabolite control protein A (CcpA) of Bacillus stearothermophilus No. 236, a strong xylanolytic bacterium, was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the ccpA gene corresponded to an open reading frame of 1,005 bp that encodes a polypeptide of 334 amino acid residues with a calculated molecular mass of 36,902 kDa. The CcpA protein belonging to the LacI/GalR family of transcriptional regulators was produced by a recombinant E. coli strain expressing the B. stearothermophilus No. 236 ccpA gene and purified to apparent homogeneity. The transcription start site was mapped at a position 63 nucleotides upstream of the translation initiation codon, and a presumed promoter sequence was also identified. The deduced amino acid sequence of the ccpA gene product contained the helix-turn-helix motif found in many DNA-binding proteins, and showed the highest identity (62%) with CcpA from B. subtilis. The B. stearothermophilus No. 236 ccpA gene was demonstrated to be able to complement a B. subtilis ccpA mutant that exhibited two distinct mutant phenotypes: a growth defect and a release of carbon catabolite repression (CCR). These results indicate that the ccpA gene product of B. stearothermophilus No. 236 is functionally active also in B. subtilis. Electrophoretic mobility shift assay with the purified CcpA revealed that the CcpA of B. stearothermophilus No. 236 bound specifically to the xynA creB (catabolite responsive element B) sequence. Taken together, these results strongly suggest that the CcpA protein participates in CCR of B. stearothermophilus No. 236 xynA gene.