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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

Schematic process for high-throughput screening of translational fusion partners (TFPs) for various target proteins.The 3′-end truncated cDNA library was constructed in YGaINV vector containing mature invertase gene (mSUC2). TFPs accelerating secretion of invertase were collected from a cDNA library and transferred to an in vivo recombination vector, containing a linker (black bar) and a defective invertase gene (dSUC2) to construct a TFP library, YGa-TFP-dV45. Target genes were flanked with the linker and N-terminal invertase fragments by overlap extension PCR and transformed with the SwaI linearised TFP library.
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f4: Schematic process for high-throughput screening of translational fusion partners (TFPs) for various target proteins.The 3′-end truncated cDNA library was constructed in YGaINV vector containing mature invertase gene (mSUC2). TFPs accelerating secretion of invertase were collected from a cDNA library and transferred to an in vivo recombination vector, containing a linker (black bar) and a defective invertase gene (dSUC2) to construct a TFP library, YGa-TFP-dV45. Target genes were flanked with the linker and N-terminal invertase fragments by overlap extension PCR and transformed with the SwaI linearised TFP library.

Mentions: Although the TFP trap system is powerful for the selection of an optimal TFP for a target protein, high-throughput application of this technology for many target proteins is restricted by the requirement for recursive construction of a library. To solve this problem and further confirm the TFP trap technology, we constructed a TFP library that could be easily applicable to a large number of target proteins. About 3 × 103 TFPs secreting invertase were collected from the 3′-end truncated cDNA library in YGa-TFP-INV, which contained TFPs for mature invertase (mSUC2) secretion (Fig. 4). To reduce the background level of transformants on sucrose media, SfiI-digested TFP fragments of YGa-TFP-INV were subcloned into YGa-TFP-dV45 containing a defective SUC2 (dSUC2) in which the N-terminal 45 aa were deleted (Fig. 4). Test transformation of the YGa-TFP-dV45 library into invertase-deficient S. cerevisiae produced thousands of transformants on UD medium, but no transformants on sucrose medium. Functional invertase could be recovered only when the SwaI-linearised YGa-TFP-dV45 plasmid containing the TFP library and a target gene flanked with the linker and N-terminal parts of invertase were co-transformed into yeast, and the circular plasmids were reconstructed by in vivo recombination (Fig. 4). The hIL2 gene was used again as a model target protein to screen for optimal TFPs. Overlap extension PCR was performed to flank the hIL2 gene with linker DNA and a 0.3-kb DNA fragment encoding the N-terminal part of invertase at the 5′ and 3′ ends, respectively. Then, the SwaI-digested YGa-TFP-dV45 plasmid containing the TFP library and the insert fragment were co-transformed into invertase-deficient S. cerevisiae Y2805Δgal80Δsuc2. Correct insertion of the target gene between an optimal TFP rendering secretion of hIL-2 and dSUC2 by in vivo recombination supported the growth of transformants on sucrose medium. Approximately, 2 × 104 transformants were formed on UD medium, whereas 24 transformants were obtained on sucrose medium.


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)

Schematic process for high-throughput screening of translational fusion partners (TFPs) for various target proteins.The 3′-end truncated cDNA library was constructed in YGaINV vector containing mature invertase gene (mSUC2). TFPs accelerating secretion of invertase were collected from a cDNA library and transferred to an in vivo recombination vector, containing a linker (black bar) and a defective invertase gene (dSUC2) to construct a TFP library, YGa-TFP-dV45. Target genes were flanked with the linker and N-terminal invertase fragments by overlap extension PCR and transformed with the SwaI linearised TFP library.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Schematic process for high-throughput screening of translational fusion partners (TFPs) for various target proteins.The 3′-end truncated cDNA library was constructed in YGaINV vector containing mature invertase gene (mSUC2). TFPs accelerating secretion of invertase were collected from a cDNA library and transferred to an in vivo recombination vector, containing a linker (black bar) and a defective invertase gene (dSUC2) to construct a TFP library, YGa-TFP-dV45. Target genes were flanked with the linker and N-terminal invertase fragments by overlap extension PCR and transformed with the SwaI linearised TFP library.
Mentions: Although the TFP trap system is powerful for the selection of an optimal TFP for a target protein, high-throughput application of this technology for many target proteins is restricted by the requirement for recursive construction of a library. To solve this problem and further confirm the TFP trap technology, we constructed a TFP library that could be easily applicable to a large number of target proteins. About 3 × 103 TFPs secreting invertase were collected from the 3′-end truncated cDNA library in YGa-TFP-INV, which contained TFPs for mature invertase (mSUC2) secretion (Fig. 4). To reduce the background level of transformants on sucrose media, SfiI-digested TFP fragments of YGa-TFP-INV were subcloned into YGa-TFP-dV45 containing a defective SUC2 (dSUC2) in which the N-terminal 45 aa were deleted (Fig. 4). Test transformation of the YGa-TFP-dV45 library into invertase-deficient S. cerevisiae produced thousands of transformants on UD medium, but no transformants on sucrose medium. Functional invertase could be recovered only when the SwaI-linearised YGa-TFP-dV45 plasmid containing the TFP library and a target gene flanked with the linker and N-terminal parts of invertase were co-transformed into yeast, and the circular plasmids were reconstructed by in vivo recombination (Fig. 4). The hIL2 gene was used again as a model target protein to screen for optimal TFPs. Overlap extension PCR was performed to flank the hIL2 gene with linker DNA and a 0.3-kb DNA fragment encoding the N-terminal part of invertase at the 5′ and 3′ ends, respectively. Then, the SwaI-digested YGa-TFP-dV45 plasmid containing the TFP library and the insert fragment were co-transformed into invertase-deficient S. cerevisiae Y2805Δgal80Δsuc2. Correct insertion of the target gene between an optimal TFP rendering secretion of hIL-2 and dSUC2 by in vivo recombination supported the growth of transformants on sucrose medium. Approximately, 2 × 104 transformants were formed on UD medium, whereas 24 transformants were obtained on sucrose medium.

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