RESUMO
Signal peptides (SPs) are one of the most important factors for suitable secretion of the recombinant heterologous proteins in Escherichia coli (E. coli). The objective of this study was to identify a panel of signal peptides (among the 90 biologically active SPs) required for the secretory production of interferon-beta 1b (IFN-beta 1b) recombinant protein into the periplasmic space of E. coli host. In the initial step, after predicting the accurate locations of the cleavage sites of signal peptides and their discrimination scores using SignalP 4.1 server, 31 SPs were eliminated from further analysis because their discrimination scores were less than 0.5 or their cleavage sites were inappropriately located. Therefore, only 59 SPs could be theoretically applied to secrete IFN-beta 1b into the periplasmic space of E. coli. The physico-chemical and the solubility properties, which are necessary parameters for selecting appropriate SPs, were predicted using ProtParam and SOLpro servers using the 59 remaining signal peptides. The final subcellular localization of IFN-beta 1b in combination with different SPs was predicted using ProtComB server. Consequently, according to the ranking of 59 confirmed SPs, the obtained results revealed that SPs Flagellar P-ring protein (flgI), Glucan 1,3-beta-glucosidase I/II (EXG1) and outer membrane protein C (OmpC) were theoretically the most potent and desirable SPs for secretion of recombinant IFN-beta 1b into the periplasmic space of E. coli. For further studies in the future, the experimental investigations on the obtained results will be considered.
Assuntos
Biotecnologia , Escherichia coli/metabolismo , Interferon beta-1b/biossíntese , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Transporte Biológico , Simulação por Computador , Interferon beta-1b/genética , Proteínas Recombinantes/genéticaRESUMO
Recombinant interferon-ß1b (IFN-ß1b) is an effective remedy against multiple sclerosis and other diseases. However, use of small polypeptide (molecular weight is around 18.5 kDa) is limited due to poor solubility, stability, and short half-life in systemic circulation. To solve this problem, we constructed two variants of PASylated IFN-ß1b, with PAS sequence at C- or N-terminus of IFN-ß1b. The PAS-modified proteins demonstrated 4-fold increase in hydrodynamic volume of the molecule combined with 2-fold increase of in vitro biological activity, as well as advanced stability and solubility of the protein in solution as opposed to unmodified IFN-ß1b. Our results demonstrate that PASylation has a positive impact on stability, solubility, and functional activity of IFN-ß1b and potentially might improve pharmacokinetic properties of the molecule as a therapeutic agent.
Assuntos
Fatores Imunológicos/metabolismo , Interferon beta-1b/genética , Interferon beta-1b/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Meia-Vida , Humanos , Fatores Imunológicos/genética , Fatores Imunológicos/uso terapêutico , Interferon beta-1b/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Estabilidade Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , SolubilidadeRESUMO
A new solubilization method of recombinant interferon beta-1b (IFNß-1b) from the inclusion bodies was developed. This method allows to extract the target protein selectively in the solutions of different alcohols, such as ethanol, propanol and isopropanol. It was shown that the more effective IFNß-1b solubilization was achieved in the 55% propanol solution. This method allowed to extract the target protein from inclusion bodies around 85-90%, and significantly reduced Escherichia coli content in the solubilizate, in comparison with standard methods.
