Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris

Abstract

The expression of heterologous proteins may exert severe stress on the host cells at different levels. Depending on the specific features of the product, different steps may be rate-limiting. For the secretion of recombinant proteins from yeast cells, folding and disulfide bond formation were identified as rate-limiting in several cases and the induction of the chaperone BiP (binding protein) is described. During the development of Pichia pastoris strains secreting human trypsinogen, a severe limitation of the amount of secreted product was identified. Strains using either the AOX1 or the GAP promoter were compared at different gene copy numbers. With the constitutive GAP promoter, no effect on the expression level was observed, whereas with the inducible AOX1 promoter an increase of the copy number above two resulted in a decrease of expression. To identify whether part of the product remained in the cells, lysates were fractionated and significant amounts of the product were identified in the insoluble fraction containing the endoplasmic reticulum, while the soluble cytosolic fraction contained product only in clones using the GAP promoter. An increase of BiP was observed upon induction of expression, indicating that the intracellular product fraction exerts an unfolded protein response in the host cells. A strain using the GAP promoter was grown both on glucose and methanol and trypsinogen was identified in the insoluble fractions of both cultures, but only in the soluble fraction of the glucose grown cultures, indicating that the amounts and distribution of intracellularly retained product depends on the culture conditions, especially the carbon source.

Heterologous protein production using the Pichia pastoris expression system

“…In contrast to the work described above, where various researchers found a direct correlation between gene copy number and subsequent yield and activity of the gene product, Hohenblum et al [47] did not note any increase in the expression of a recombinant human trypsinogen when the gene dosage was increased from one to three copies under the control of the GAP promoter. However, an increase in the expression level of this protein was observed using the AOX1 promoter when the gene copy number was increased to two, but levels of expression fell upon further copy number increases.…”

“…One of the advantages of this phenotype is that low growth rates may be desirable for production of certain recombinant products [26,27]. Currently, the majority of researchers use the Mut + phenotype [47,102,129], although some researchers are also using the Mut s phenotype [1,71,83,85,126].…”

“…However, increased copy number might reasonably be expected to exert a knock-on effect on transcription and translation, both of which may become rate-limiting due to a lack of resources, such as precursors and energy [47], as has been previously reported for recombinant protein production in E. coli [62,93]. However, in Pichia it has been proposed that it is more likely that any limitations are due to post-translational events, such as folding within the endoplasmic reticulum (ER), membrane translocation and signal sequence processing [47].…”

“…However, in Pichia it has been proposed that it is more likely that any limitations are due to post-translational events, such as folding within the endoplasmic reticulum (ER), membrane translocation and signal sequence processing [47]. Sunga and Cregg [107] also demonstrated that increasing the copy number of a β-galactosidase (β-gal) gene (lacZ ), under the control of the FLD1 promoter, increased the relative activity of the enzyme proportionately.…”

“…correct folding, disulphide bond formation, O-Heterologous protein production using Pichia pastoris expression system 257 and N -linked glycosylation and processing of signal sequences. Folding and disulphide bond formation have been identified in some cases as the 'rate-limiting' step in the production of foreign proteins from Pichia [47], i.e. the ability of the organism to process, fold and secrete the recombinant products determines the productivity of the Pichia expression system.…”