Improved Secretion of Native Human Insulin-Like Growth Factor 1 from gas1 Mutant Saccharomyces cerevisiae Cells

Abstract

We studied the secretion of recombinant human insulin-like growth factor 1 (rhIGF-1) from transformed yeast cells. The hIGF-1 gene was fused to the mating factor ␣ prepro-leader sequence under the control of the constitutive ACT1 promoter. We found that the inactivation of the GAS1 gene in the host strain led to a supersecretory phenotype yielding a considerable increase, from 8 to 55 mg/liter, in rhIGF-1 production.

High-level soluble expression of hIGF-1 fusion protein in recombinant Escherichia coli

“…On the contrast, the expression level of hIGF-1 in yeast was as low as only 55 mg/L [6]. Similarly, the production of hIGF-1 in several transgenic plant and animal systems was also very low.…”

“…Up to now, a variety of expression systems have been established to produce hIGF-1, including Escherichia coli [1,3,4], yeast [5,6], cell-free system [7], transgenic plants, and rabbits [2,8,9]. The comparison of hIGF-1 expression in different hosts was summarized in Table 1.…”

Random and direct mutagenesis to enhance protein secretion inAshbya gossypii

“…GAS1 was chosen as a target for A. gossypii strain improvement because in yeast it encodes a cross-linking enzyme known to affect the structure and permeability of the cell wall. Cell wall defective strains deleted in the GAS1 gene of S. cerevisiae , 29 P. pastoris 30 and Z. bailii 31 have been reported as super-secreting. Deletion of GAS1 in S. cerevisiae resulted in an almost 7-fold increase in the level of human hIGF1 29 .…”

“…Cell wall defective strains deleted in the GAS1 gene of S. cerevisiae , 29 P. pastoris 30 and Z. bailii 31 have been reported as super-secreting. Deletion of GAS1 in S. cerevisiae resulted in an almost 7-fold increase in the level of human hIGF1 29 . Similarly, secreted enzymatic activity was almost doubled when GAS1 was deleted in Z. bailii producing C. rugosa lipase Crl1p or Y. lipolytica protease Xpr2p 31 .…”

“…Mutations in genes involved in the construction and in the maintenance of the cell wall, such as PMR1 , SEC14 , ERD1 , MNN9 and MNN10 , have in some cases been demonstrated to lead to supersecreting mutants in S. cerevisiae and other yeast 28 . In addition, the deletion of the cell wall cross-linking enzyme glycophospholipid-anchored surface Gas1p led to an almost 7-fold increase in the level of human insulin-like growth factor 1 (hIGF1) in S. cerevisiae 29 . In Pichia pastoris , deletion of the GAS1 homolog resulted in a supersecreting phenotype for the heterologou s Rhizopus oryzae lipase, but had no effect on the secretion of recombinant human trypsinogen and albumin 30 .…”