The flhD and flhC genes constitute the flagellar master operon whose products are required for expression of all the remaining flagellar operons in Salmonella typhimurium. Here we report the molecular structure and in vivo and in vitro expression of the flhD operon. Nucleotide sequence analysis revealed that the upstream region of this operon contains the consensus sequence for the cAMP-CRP binding site. Primer extension analysis demonstrated six possible transcription start sites for this operon. They include CRP-dependent and CRP-repressible transcription start sites. The CRP-dependent transcription start site is located 203 bp upstream of the initiation codon of the flhD gene and preceded by the consensus sequences of the -10 and -35 regions of the σ 70 -dependent promoter. The putative cAMP-CRP binding site is located centered 70 bp upstream of this start site. The CRP-repressible transcription start site is located within this putative cAMP-CRP binding site. These two start sites were confirmed by in vitro transcription experiments using σ 70 -RNA polymerase with or without cAMP-CRP.

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To achieve quality control of therapeutic proteins, it is important to compare their biological potency with their clinical effects.1) Therefore, an animal-based assay is preferred for measuring their bioactivity. However, the use of laboratory animals poses ethical concerns, is time consuming and requires skilled staff and sophisticated experimental facilities. Furthermore, in vivo bioassays have low precision and accuracy, and hence, there is a need for an alternative assay method to estimate in vivo bioactivity.To date, attempts have been made to replace traditional bioassays with physicochemical techniques for potency determination of insulin and growth hormone because it is possible to design and validate manufacturing processes in the production of properly folded materials.2,3) Potency of these pharmaceutical preparations can be calculated using the content assay with HPLC, which quantifies the peak area of a product against that of a reference standard of known potency. The results of the HPLC assay have been shown to correlate well with those of the bioassay.2,3) In addition, the HPLC assay is rapid, precise and less labour intensive than the bioassay. However, for some glycoproteins, alternative assay methods using a quantitative charge-based separation technique, such as isoelectric focusing (IEF) and capillary zone electrophoresis (CZE), have been shown to be feasible for estimating in vivo bioactivity 4,5) because negatively charged sialic acid residues are critical for in vivo bioactivity. For example, Mulders et al. 4) reported that quantitative assessment of the isoform distribution by IEF may provide a method for predicting in vivo biological potency of preparations containing recombinant human follicle-stimulating hormone (rhFSH). Close correlation between results of experimental in vivo bioactivities and IEF-predicted in vivo bioactivities suggest that the only factor affecting prediction of biological potency is the result of quantitative isoform distribution in rhFSH.Erythropoietin (EPO) is a glycoprotein hormone that regulates the red blood cell level by stimulating maturation of erythroid precursor cells. 6) Recombinant human EPO (rhEPO) produced in Chinese hamster ovary (CHO) cells is extensively used for serve anaemia therapy.7) EPO comprises a 165-amino acid protein with 40% of its molecular weight accounted for by carbohydrates.8,9) EPO contains three Nglycans located on Asn residues at positions 24, 38, and 83 and one mucin-type O-glycan located on the Ser-126 residue. The N-linked carbohydrates comprise bi-, tri-and tetraantennary oligosaccharides, which typically terminate with a negatively charged sialic acid residue. 10,11) Because of the variable number of sialic acid residues, the product of EPO usually exists as a mixture of isoforms with different isoelectric points. Several studies have reported that sialic acid residues are critical for in vivo bioactivity of EPO.12,13) Isoforms having a higher sialic acid content are seen to display higher in vivo bioactivity, longer seru...