Carbon source feeding strategies for recombinant protein expression in Pichia pastoris and Pichia methanolica

Pichia pastoris and Pichia methanolica have been used as expression systems for the production of recombinant protein. The main problems of the production are the slow hierarchic consumption of ethanol and acetate which cause toxicity problems due to methanol accumulation when this surpasses 0.5 gl-1. In some cases, the laboratory scale cultures does not show the methanol accumulation problems because the cells are usually washed before changing the depleted carbon source culture media, by a fresh one, supplemented with methanol; a strategy that is clearly inapplicable in the industrial productions. Other authors use to feed the methanol before the depletion of glycerol or Dglucose, but this practice does not guaranty the ethanol and acetate fast consumption; leading to methanol accumulation and toxicity problems. It was concluded that pre-induction stage strategy should be studied in detail and that it is very important to start the induction stage with a concentration of biomass as great as possible. On the other side, it is essential that there should be a monitoring of ethanol and acetate until reaching non-toxic methanol stable-concentration and these conditions should be maintained till the end of the process

Laboratory scale production of the human recombinant iduronate 2-sulfate sulfatase-Like from Pichia pastoris

Clone IDS28 of the yeast Pichia pastoris expressing the human iduronate 2-sulfate sulfatase-Like (hIDSLike) was employed for low-scale production of the recombinant enzyme in a saline culture media without phosphate. The biological activity found was between 7.3 and 29.5 nmol h-1 mg-1 of total protein. It is about 1.73 to 7 times higher than the result obtained with the same clone in shake flask culture

Cloning and shake flask expression of hrIDS-Like in Pichia pastoris

The human Iduronate-2-sulfate sulfatase (hIDS-Like) was cloned into the methylotrophic yeast Pichia pastoris under the control of alcohol oxidase promoter (AOX1) and the -mating factor signal peptide (a-factor). Six clones were identified by PCR. Using clone IDS28, the enzyme was secreted into the culture medium, yielding a protein with an activity of 4.213 nmol.h-1.mg of total protein-1 at 72 h, in 0.5% v/v methanol. Several bands were revealed by western-blot, indicating that a P. pastoris processing was slightly different than in mammalian cells