Enhanced recombinant human growth hormone production by synchronous populations of two different strains of pichia pastoris under GAP promoter

Hüccetoğulları, Damla
Cell cycle synchronization used as a common procedure for understanding various genomic and metabolic phenomena in a cell is also an essential tool to optimize the production of biomolecules. In this work, the effect of cell cycle synchronization on the recombinant human growth hormone (rhGH) production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was investigated. Starvation as a cost effective and readily applicable synchronization method was performed to investigate various cell cultures at age of OD600 = 4, 8, and 16 to determine the most productive strategy. Thus, two different experimental setups were used, one of which was in air-filtered shake bioreactor cultures to consider the impact of semi-defined and complex pre-cultivation media on the efficiency of starvation concerned with the cell morphology and budding index. The second setup, a 3.0 L-bioreactor, was used to compare the recombinant protein (r-protein) productions in both synchronous and asynchronous populations grown to OD600 = 4, 8, and 16 by using exponential glucose feeding with pre-determined specific growth rate of μ0 = 0.10 h-1. The highest rhGH concentration was obtained from synchronous cell population at age of OD600 = 16 as CrhGH = 570 mg L-1 at t = 15 h where the cell concentration was CX = 100 g L-1. The overall product and cell yield on total substrate were obtained as Y’P/S = 2.06 mg g-1 and Y’X/S = 0.13 g g-1, respectively. We conclude that, this work demonstrates the impact of using aged synchronous populations of P. pastoris in r-protein production and paves the way for the design of the starvation based fed-batch operations to increase r-protein production by P. pastoris. 


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Citation Formats
D. Hüccetoğulları, “Enhanced recombinant human growth hormone production by synchronous populations of two different strains of pichia pastoris under GAP promoter,” M.S. - Master of Science, Middle East Technical University, 2016.