RESUMEN
In this research it was attempted to overexpress the acidic subunit, from the 11S amaranth seed globulin termed amarantin, modified with antihypertensive peptides in Escherichia coli Rosetta (DE3) by manipulating some factors in batch fermenter such as growth medium composition, inducer (isopropyl ß-D-thiogalactopyranoside [IPTG] or lactose), air flow, cultivation temperature, agitation speed and induction time. The possibility of using several minimal media and lactose as inducer to increase yields of the recombinant protein was investigated. Previous fermentations at flask level showed that two minimal culture media (A6 and A7) and 0.5% (w/v) lactose presented high yields of the engineered protein expression. Thus, the latter two media were tested at fermenter level, the lactose inducer, and different environmental conditions. Factors with significant effects were identified by Plackett-Burman design with center points and were adjusted at the level suggested and the yields of the recombinant protein were increased from 303.2 to 1,531 mg L(-1) in A6 and from 363.4 to 1,681 mg L(-1) in A7. Unlike some patents where the highest productivity was achieved at 24 h or afterwards, in this research the best productivity of the recombinant acidic subunit was attained at 4 and 6 h of induction using both media, respectively.
Asunto(s)
Amaranthus/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Globulinas/biosíntesis , Lactosa/metabolismo , Proteínas de Plantas/biosíntesis , Reactores Biológicos , Biotecnología/métodos , Fermentación , Globulinas/química , Globulinas/genética , Patentes como Asunto , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The expression in Escherichia coli strain Rosetta of the recombinant acidic subunit from the 11S amaranth seed storage protein (protein ACM3) was studied at flask and at bioreactor levels. This subunit was modified by inserting four Val-Tyr antihypertensive peptides in tandem into its third variable region and also with the tripeptide Ile-Pro-Pro in the Cterminal region. Flasks experiments allowed us to define the best conditions for the preparation and expression and accumulation of the protein ACM3, including the certainty of its presence within the cells especially as an insoluble fraction. The effects of cultivation temperature, aeration rate and agitation speed on the production of the protein ACM3 was tested in a 5-L batch bioreactor. Applying response surface methodology (RSM) we found that the aeration rate was the most significant factor affecting in a positively way the production yields and productivity of the recombinant protein. Temperature had effect only in conjunction to aeration. The highest recombinant acidic subunit concentration (747 mg L-1) and the highest productivity (186 mg L-1 h-1) were attained in 4 h of cultivation when the factors evaluated were controlled at its central values: 0.1 vvm, 300 rpm, and 30.5° C. Results from this study indicate that RSM is an effective technique to maximize the production of this recombinant protein.