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An Efficient Genome-Wide Fusion Partner Screening System for Secretion of Recombinant Proteins in Yeast.

Bae JH, Sung BH, Kim HJ, Park SH, Lim KM, Kim MJ, Lee CR, Sohn JH - Sci Rep (2015)

Bottom Line: Optimal TFPs for secretion of hIL-2 and hIL-32 were easily selected, yielding secretion of these proteins up to hundreds of mg/L.Selected TFPs were found to be useful for the hypersecretion of other recombinant proteins at yields of up to several g/L.This screening technique could provide new methods for the production of various types of difficult-to-express proteins.

View Article: PubMed Central - PubMed

Affiliation: Bioenergy and Biochemical Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea.

ABSTRACT
To produce rarely secreted recombinant proteins in the yeast Saccharomyces cerevisiae, we developed a novel genome-wide optimal translational fusion partner (TFP) screening system that involves recruitment of an optimal secretion signal and fusion partner. A TFP library was constructed from a genomic and truncated cDNA library by using the invertase-based signal sequence trap technique. The efficiency of the system was demonstrated using two rarely secreted proteins, human interleukin (hIL)-2 and hIL-32. Optimal TFPs for secretion of hIL-2 and hIL-32 were easily selected, yielding secretion of these proteins up to hundreds of mg/L. Moreover, numerous uncovered yeast secretion signals and fusion partners were identified, leading to efficient secretion of various recombinant proteins. Selected TFPs were found to be useful for the hypersecretion of other recombinant proteins at yields of up to several g/L. This screening technique could provide new methods for the production of various types of difficult-to-express proteins.

No MeSH data available.


Related in: MedlinePlus

Invertase-based translational fusion partner trap system.(a) Growth patterns of Saccharomyces cerevisiae Y2805 Δsuc2 recombinant strains carrying the indicated plasmids on different carbon sources, glucose and sucrose. (b) Schematic representation of the genetic screening system for the cloning of optimal translational fusion partners (TFPs) to accelerate secretion of target proteins (X).
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f1: Invertase-based translational fusion partner trap system.(a) Growth patterns of Saccharomyces cerevisiae Y2805 Δsuc2 recombinant strains carrying the indicated plasmids on different carbon sources, glucose and sucrose. (b) Schematic representation of the genetic screening system for the cloning of optimal translational fusion partners (TFPs) to accelerate secretion of target proteins (X).

Mentions: Secretion of invertase, encoded by SUC2, is prerequisite for yeast cell growth on media containing sucrose as a sole carbon source. The growth defect of the invertase deletion mutant S. cerevisiae Y2805Δgal80Δsuc2 on sucrose medium was recovered by functional complementation of the SUC2 gene (Fig. 1a). Interestingly, we found that invertase exhibited different fates when it was directly fused to HSA or hIL-2. The expression of the HSA-SUC2 fusion protein supported growth of the invertase mutant strain on sucrose medium, whereas the expression of hIL2-SUC2 did not support growth on sucrose medium. Therefore, fusion of the well-secreted HSA protein did not disturb invertase secretion. On the other hand, fusion of the rarely secreted hIL-2 protein completely blocked the secretion of invertase. This feature of invertase fused with a low-secretion-competent protein (X) like hIL-2 made it possible to design a genetic trap for target protein-specific TFPs from a fusion partner library derived from truncated cDNAs and genomic DNA (Fig. 1b).


An Efficient Genome-Wide Fusion Partner Screening System for Secretion of Recombinant Proteins in Yeast.

Bae JH, Sung BH, Kim HJ, Park SH, Lim KM, Kim MJ, Lee CR, Sohn JH - Sci Rep (2015)

Invertase-based translational fusion partner trap system.(a) Growth patterns of Saccharomyces cerevisiae Y2805 Δsuc2 recombinant strains carrying the indicated plasmids on different carbon sources, glucose and sucrose. (b) Schematic representation of the genetic screening system for the cloning of optimal translational fusion partners (TFPs) to accelerate secretion of target proteins (X).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4508530&req=5

f1: Invertase-based translational fusion partner trap system.(a) Growth patterns of Saccharomyces cerevisiae Y2805 Δsuc2 recombinant strains carrying the indicated plasmids on different carbon sources, glucose and sucrose. (b) Schematic representation of the genetic screening system for the cloning of optimal translational fusion partners (TFPs) to accelerate secretion of target proteins (X).
Mentions: Secretion of invertase, encoded by SUC2, is prerequisite for yeast cell growth on media containing sucrose as a sole carbon source. The growth defect of the invertase deletion mutant S. cerevisiae Y2805Δgal80Δsuc2 on sucrose medium was recovered by functional complementation of the SUC2 gene (Fig. 1a). Interestingly, we found that invertase exhibited different fates when it was directly fused to HSA or hIL-2. The expression of the HSA-SUC2 fusion protein supported growth of the invertase mutant strain on sucrose medium, whereas the expression of hIL2-SUC2 did not support growth on sucrose medium. Therefore, fusion of the well-secreted HSA protein did not disturb invertase secretion. On the other hand, fusion of the rarely secreted hIL-2 protein completely blocked the secretion of invertase. This feature of invertase fused with a low-secretion-competent protein (X) like hIL-2 made it possible to design a genetic trap for target protein-specific TFPs from a fusion partner library derived from truncated cDNAs and genomic DNA (Fig. 1b).

Bottom Line: Optimal TFPs for secretion of hIL-2 and hIL-32 were easily selected, yielding secretion of these proteins up to hundreds of mg/L.Selected TFPs were found to be useful for the hypersecretion of other recombinant proteins at yields of up to several g/L.This screening technique could provide new methods for the production of various types of difficult-to-express proteins.

View Article: PubMed Central - PubMed

Affiliation: Bioenergy and Biochemical Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea.

ABSTRACT
To produce rarely secreted recombinant proteins in the yeast Saccharomyces cerevisiae, we developed a novel genome-wide optimal translational fusion partner (TFP) screening system that involves recruitment of an optimal secretion signal and fusion partner. A TFP library was constructed from a genomic and truncated cDNA library by using the invertase-based signal sequence trap technique. The efficiency of the system was demonstrated using two rarely secreted proteins, human interleukin (hIL)-2 and hIL-32. Optimal TFPs for secretion of hIL-2 and hIL-32 were easily selected, yielding secretion of these proteins up to hundreds of mg/L. Moreover, numerous uncovered yeast secretion signals and fusion partners were identified, leading to efficient secretion of various recombinant proteins. Selected TFPs were found to be useful for the hypersecretion of other recombinant proteins at yields of up to several g/L. This screening technique could provide new methods for the production of various types of difficult-to-express proteins.

No MeSH data available.


Related in: MedlinePlus