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. 2017 Fall;16(4):1555-1564.

Evaluation of Sorbitol-Methanol Co-Feeding Strategy on Production of Recombinant Human Growth Hormone in Pichia Pastoris

Saeed Azadi  1 , Arash Mahboubi  2   3 , Nasser Naghdi  4 , Roya Solaimanian  5 , Seyyed Alireza Mortazavi  1   3
Affiliations

Evaluation of Sorbitol-Methanol Co-Feeding Strategy on Production of Recombinant Human Growth Hormone in Pichia Pastoris

Saeed Azadi et al. Iran J Pharm Res. 2017 Fall.

Abstract

Recombinant protein production in Pichia pastoris is based on alcohol oxidase promoters which are regulated by methanol. However, the use of methanol has several disadvantages, which is why current trends in bioprocess development with Pichia pastoris (P. pastoris) are focusing on methanol mixed feeding strategies. This work aimed to develop a new experimental method and compare the effect of various fractions of sorbitol in mixed feeding strategy with stepwise addition of methanol to maximize the productivity of human growth hormone. Accordingly, fed-batch culture performed with a mixed feed of sorbitol/methanol. Sorbitol at concentrations of 30, 40, 50 and 60 gram per liter was added in batch-wise mode to the medium at the beginning of induction phase and the rate of methanol addition was increased stepwise during the first 12 h of production and then kept constant. In order to understand the effects of various co-substrate feeding strategies on P. pastoris and its production of hGH, cell mass, dry cell weight, total protein, hGH expression level and hGH concentration were analyzed and the results compared with the basic feeding protocol using one-way analysis of variance (ANOVA). According to the obtained results, sorbitol at a concentration of 50 g/L could significantly increase the production yield. Under such optimal conditions cell biomass was 108 g/L (DCW), total protein was 0.807 g/L, expression level was 83.1% and rhGH concentration was 0.667 g/L following 30 h induction.

Keywords: Fermentation; Mixed-feed strategy; Pichia Pastoris; Sorbitol.

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Figures

Figure 1
Figure 1
The effect of various sorbitol substrate concentration on cell density
Figure 2
Figure 2
The effect of different concentrations of sorbitol on total protein determined using Bradford protein assay.
Figure 3
Figure 3
The effect of various concentration of sorbitol on the level of protein expression (percent
Figure 4
Figure 4
Protein expression and densitometric analysis of three fermentation runs at concentration of 50 g/L
Figure 5
Figure 5
The effect of sorbitol concentration on rhGH production
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