RESUMO
The Gram-positive bacterium Bacillus megaterium was systematically developed for the plasmid-based production of recombinant proteins at the gram-per-liter scale. The amount of protein produced per cell was found strongly correlated to the codon usage of the heterologous gene of interest in comparison to the codon usage of B. megaterium. For analyzing the influence of rare codons on the translational efficiency and protein production in B. megaterium, a test system using the gene for the green fluorescent protein (GFP) as reporter was established. For this purpose, four consecutive identical codons were introduced into the 5' end of gfp and the resulting variations in GFP formation were quantified. Introduction of the rare codons GCC, CGG, and ACC for alanine, arginine, and threonine reduced GFP production 2.1-, 3.3-, and 1.7-fold in comparison to the favored codons GCU, CGU, and ACA, respectively. Coexpression of the corresponding rare codon tRNA (rctRNA) genes improved GFP production 4.2-, 2.7-, and 1.7-fold, respectively. The system was applied to the production of a formate dehydrogenase (FDH) from Mycobacterium vaccae and an extracellular hydrolase (TFH) from Thermobifida fusca. Coexpression of one to three different rctRNA genes resulted in an up to 18-fold increased protein production. Interestingly, rctRNA gene coexpression also elevated the production of M. vaccae FDH and T. fusca TFH from codon optimized genes, indicating a general positive effect by rctRNA gene overexpression on the protein production in B. megaterium. Thus, the basis for a B. megaterium enhanced production strain coexpressing rctRNA genes was laid.
Assuntos
Bacillus megaterium/metabolismo , Códon , Engenharia Metabólica/métodos , Biossíntese de Proteínas , Engenharia de Proteínas/métodos , RNA de Transferência/metabolismo , Proteínas Recombinantes/biossíntese , Actinobacteria/enzimologia , Actinobacteria/genética , Bacillus megaterium/genética , Formiato Desidrogenases/genética , Formiato Desidrogenases/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Hidrolases/genética , Hidrolases/metabolismo , Mycobacterium/enzimologia , Mycobacterium/genética , RNA de Transferência/genética , Proteínas Recombinantes/genéticaRESUMO
Urothelial carcinoma of the urinary bladder (UCB) or bladder cancer remains a major health problem with high morbidity and mortality rates, especially in the western world. UCB is also associated with the highest cost per patient. In recent years numerous markers have been evaluated for suitability in UCB detection and surveillance. However, to date none of these markers can replace or even reduce the use of routine tools (cytology and cystoscopy). Our current study described UCB's extensive expression profile and highlighted the variations with normal bladder tissue. Our data revealed that JUP, PTGDR, KLRF1, MT-TC, and RNU6-135P are associated with prognosis in patients with UCB. The microarray expression data identified also S100A12, S100A8, and NAMPT as potential UCB biomarkers. Pathway analysis revealed that natural killer cell mediated cytotoxicity is the most involved pathway. Our analysis showed that S100A12 protein may be useful as a biomarker for early UCB detection. Plasma S100A12 has been observed in patients with UCB with an overall sensitivity of 90.5% and a specificity of 75%. S100A12 is highly expressed preferably in high-grade and high-stage UCB. Furthermore, using a panel of more than hundred urine samples, a prototype lateral flow test for the transcription factor Engrailed-2 (EN2) also showed reasonable sensitivity (85%) and specificity (71%). Such findings provide confidence to further improve and refine the EN2 rapid test for use in clinical practice. In conclusion, S100A12 and EN2 have shown potential value as biomarker candidates for UCB patients. These results can speed up the discovery of biomarkers, improving diagnostic accuracy and may help the management of UCB.