RESUMEN
Human cardiac troponin-I (cTnI) is one of the most sensitive and specific indicators, used in the diagnosis of myocardial infarction. To produce the protein efficiently, Escherichia coli and Pichia pastoris systems were used. Initial trials for the expression in E. coli were not successful, although different expression vectors with different promoters were tested. This led us to use P. pastoris for the expression. After several trials with two different expression strains of P. pastoris, it was concluded that P. pastoris was also not an optimal expression host for cTnI. Comprehensive analysis of the expression systems indicated that an efficient expression is only possible when the gene is optimized for expression in E. coli. For this purpose, the gene was optimized in-silico, but edited manually afterwards. It was synthesized and cloned into pQE-2 vector. Expression was performed using routine experimental conditions. Thus, cTnI could be efficiently expressed from the optimized gene in E. coli. The expression and purification were practical and may be used for commercial purposes since a total yield of 25µg highly pure protein per milliliter of culture could be obtained. The protein was in its ready-to-use form for many biological applications, including as a standard in diagnostic tests and an antigen for antibody production.
Asunto(s)
Troponina I/biosíntesis , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Codón , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Pichia/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Troponina I/genética , Troponina I/aislamiento & purificaciónRESUMEN
Alginate has an extensive usage in the immobilization of many cell types. Although they have high biocompatibility, commercial alginates contain various degrees of contaminants such as polyphenols, endotoxins and proteins. Thus, these alginates show cytotoxicity against sensitive cell types such as hybridoma cells. In the studies so far, owing to this fact, commercially purchased high-priced ultrapure alginates have been used in the immobilization of hybridoma cells for monoclonal antibody production. However in this study, as a novelty, low-priced commercial alginate was purified, and then the cultivation of alginate-immobilized hybridoma cells was performed for feasible monoclonal antibody production. Low-priced commercial alginate was purified with a profitability ratio of 40%. Then, an optimized immobilization procedure was conducted effectively by using the purified alginate. During more than 25 days of cultivation, serum concentration was kept low, and approximately 2 times greater monoclonal antibody production was achieved, in comparison with its free suspended counterpart. The results showed that the efficiency of monoclonal antibody production via alginate-immobilized hybridoma cultivation can be increased by performing a proved in-house purification method. By shedding light on the efficiency of the in-house purification method, the results also indicated a feasible way of monoclonal antibody production.
Asunto(s)
Alginatos , Anticuerpos Monoclonales/biosíntesis , Hibridomas , Alginatos/química , Alginatos/aislamiento & purificación , Alginatos/toxicidad , Animales , Línea Celular Tumoral , Células Inmovilizadas/metabolismo , Ácido Glucurónico/química , Ácido Glucurónico/aislamiento & purificación , Ácido Glucurónico/toxicidad , Ácidos Hexurónicos/química , Ácidos Hexurónicos/aislamiento & purificación , Ácidos Hexurónicos/toxicidad , Ratones , Polifenoles/análisisRESUMEN
Alginate has been widely used in various applications since its first extraction. What makes this biopolymer useful is its high biocompatibility and humid gelation conditions. Both of these features bring it into prominence as an ideal immobilization material. However, there are some complicated aspects of cell immobilization using alginate biopolymers. This review discusses and clarifies these crucial points, using as an example the bioprocessing of highly fragile cells (hybridoma cells). The review focuses on the cultivation and production of alginate encapsulated cells.