RESUMO
BACKGROUND: Fibroblast growth factor 21 (FGF21) is a promising candidate for treating metabolic disorder diseases and has been used in phase II clinical trials. Currently, metabolic diseases are prevalent worldwide, underscoring the significant market potential of FGF21. Therefore, the production of FGF21 must be effectively improved to meet market demand. RESULTS: Herein, to investigate the impact of vectors and host cells on FGF21 expression, we successfully engineered strains that exhibit a high yield of FGF21. Surprisingly, the data revealed that vectors with various copy numbers significantly impact the expression of FGF21, and the results showed a 4.35-fold increase in expression levels. Furthermore, the performance of the double promoter and tandem gene expression construction design surpassed that of the conventional construction method, with a maximum difference of 2.67 times. CONCLUSION: By exploring engineered vectors and host cells, we successfully achieved high-yield production of the FGF21 strain. This breakthrough lays a solid foundation for the future industrialization of FGF21. Additionally, FGF21 can be easily, quickly and efficiently expressed, providing a better tool and platform for the research and application of more recombinant proteins.
Assuntos
Fatores de Crescimento de Fibroblastos , Vetores Genéticos , Regiões Promotoras Genéticas , Proteínas Recombinantes , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Vetores Genéticos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Expressão GênicaRESUMO
Baloxavir marboxil (BXM) is a cap-dependent nucleic acid endonuclease inhibitor, which exerts its antiviral effects after being metabolized to its active form baloxavir acid (BXA). Ethylenediamine tetra-acetic acid (EDTA) and heparin are the two most used anticoagulants in clinical blood sample collection to estimate drug levels in plasma. However, compared to heparin plasma, there is a lack of clinical pharmacokinetic data of BXA using EDTA anticoagulant tubes for blood collection. In the present study, an efficient, rapid, and sensitive ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for simultaneous quantification of BXM and its active metabolite BXA in human plasma with its isotopic baloxavir-d5 (BXA-d5) as internal standard (IS). Plasma samples (50⯵L) were undergone using acetonitrile containing 0.1â¯% formic acid a precipitant. Chromatographic separation was achieved by a Waters XBridge®C8 (2.1â¯mm × 50â¯mm, 2.5⯵m) column. The gradient mobile phase was 0.1â¯% formic acid in water (A, pH 2.8) and 0.1â¯% formic acid in acetonitrile (B) and delivered at a flow rate of 0.6â¯mL/min for 4.5â¯min. BXM and BXA were monitored using a positive electrospray triple quadrupole mass spectrometer (TRIPLE QUAD™ 6500+) via multiple reaction monitoring mode. The mass-to-charge ratios (m/z) were 572.2â247.0, 484.2â247.0 and 489.2â252.0 for BXM, BXA, and BXA-d5 (IS). Calibration curves exhibited excellent linearity in the range of 0.1-10â¯ng/mL for BXM (r2 > 0.996), and 0.3-300â¯ng/mL for BXA (r2 > 0.998). Within-run and between-run precisions in coefficients of variations were less than 11.62â¯% for BXM and less than 7.47â¯% for BXA, and accuracies in relative error were determined to be within -7.78â¯% to 5.70â¯% for BXM and -6.67â¯% to 8.56â¯% for BXA. Extraction recovery efficiency was 92.76â¯% for BXM, 95.32â¯% for BXA, and 99.26â¯% for BXA-d5, respectively. The matrix effect of BXM and BXA was in line with the requirements, where the relative deviation of the accuracy was less than 6.67â¯% and the precision was less than 6.69â¯%. The validated efficient and simple UHPLC-MS/MS method was successfully used in the pharmacokinetic study of BXM and BXA in healthy human volunteers with K2EDTA and heparin tubes for blood collection. EDTA might compete with BXA for chelating metal ions and thereby decrease the plasma ratio in whole blood, leading to approximately 50â¯% lower measurement of pharmacokinetic parameters as compared with those obtained from heparin plasma anticoagulant tubes.