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Sunday, September 13, 2009

Pichia as Protein Expression System

Pichia is a methylotrophic yeast which is widely used to express heterologous proteins using recombinant DNA technology.

Pichia pastoris (P.pastoris) is owned and licensed by Research corporation Technologies (RCT).

Pichia pastoris is a methylotrophic yeast, capable of metabolizing methanol as its sole carbon source. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde using molecular oxygen by the enzyme alcohol oxidase.

In addition to formaldehyde, this reaction generates hydrogen peroxide. To avoid hydrogen peroxide toxicity, methanol metabolism takes place within a specialized cell organelle, called the peroxisome, which sequesters toxic by-products away from the rest of the cell. Alcohol oxidase has a poor affinity for O2, and Pichia pastoris compensates by generating large amounts of the enzyme. The promoter
regulating the production of alcohol oxidase is the one used to drive heterologous protein expression in Pichia.

Alcohol Oxidase Genes
Pichia pastoris has two genes for coding alcohol oxidase AOX1 & AOX2.AOX1 is responsible for majority of the alcohol oxidase production and it is tightly regulated by methanol.

Both AOX1&AOX2 Present - Mut+ (Methanol Utilization Plus)
Only AOX2 Present - Muts (Methanol Utilization Slow)

Advantages of Pichia Expression System
  • High Cell Density
  • Genetic Manipulation is Easy
  • Efficient Protein Processing, Protein Folding & Post-Translational Modifications.
  • Faster, Easier and less expensive to use.
  • 10-100 fold higher heterologous protein expression as compared to sacchromyces cerviseae.