Abstract
The production of heterologous proteins for medical use is an important area of interest. The optimization of the bioprocesses includes the improvement of time, costs, and unit operations. Our study shows that a lectin fraction from Tepary bean (Phaseolus acutifolius) (TBLF) has cytotoxic effects on colon cancer cells and in vivo antitumorigenic activity. However, the low-yield, time-consuming, and expensive process made us focus on the development of a strategy to obtain a recombinant lectin using engineered Pichia pastoris yeast. Pure glycerol is one of the most expensive supplies; therefore, we worked on process optimization using crude glycerol from biodiesel production. Recombinant lectin (rTBL-1) production and purification were evaluated for the first time by an experimental design where crude glycerol (G65) was used and compared against pure glycerol (G99) in a controlled stirred-tank bioreactor with a fed-batch system. The recombinant lectin was detected and identified by SDS-PAGE, Western blot, and UHPLC–ESI–QTOF/MS analysis. The results show that the recombinant lectin can be produced from G65 with no significant differences with respect to G99: the reaction rates were 2.04 and 1.43 mg L−1 h−1, and the yields were 264.95 and 274.67 mgL−1, respectively. The current low cost of crude glycerol and our results show the possibility of producing heterologous proteins using this substrate with high productivity.
Highlights
Biotechnology and Biochemistry Department, Campus Irapuato, Irapuato 36821, Guanajuato, Mexico; Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C., Av
A process to obtain a recombinant lectin from the Tepary bean was developed using engineered Pichia pastoris yeast [6]
We report a novel optimization of the original process [6] using crude glycerol for high expression of the rTBL-1 by an experimental design that varies operational conditions such as pH, aeration, agitation, and temperature using engineered P. pastoris yeast
Summary
The recombinant yeast strain P. pastoris SMD1168H was provided by Dr Elaine Fitches, Department of Biosciences, Durham University, Durham DH1 3LE, UK and contains a constitutive pGAPZαB vector for lectin rTBL-1 production [6]. P. pastoris cells were grown on minimal glycerol (MG) plates containing a 1.34% (w/v) yeast nitrogen base without amino acids (YNB), 4 × 10−5 % (w/v) biotin, 2% (w/v) glycerol, and 2% (w/v) zeocin agar, and stored at 4 ◦ C. Semicrude and crude biodiesel glycerol were kindly donated by M.C. Saúl López Ordaz (Bioenergetic Laboratory, Autonomous University of Queretaro, Mexico). Crude glycerol (G65) was obtained from the transesterification reaction after biodiesel separation and neutralization with sodium hydroxide (1% w/v) followed by simple centrifugation (4000 rpm for 20 min). 4–12% (w/v) water and 30–35% (w/v) nonglycerol organic matter, pH 8.3–9.0
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