Limits...
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

Fermentation of recombinant yeast secreting hIL-32 directed by TFP10.(a) Profile of fed-batch fermentation, (b) SDS-PAGE followed by Coomassie staining analysis of fermentation broth (10 μL) retrieved at the indicated times, lane M: standard protein size marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4508530&req=5

f6: Fermentation of recombinant yeast secreting hIL-32 directed by TFP10.(a) Profile of fed-batch fermentation, (b) SDS-PAGE followed by Coomassie staining analysis of fermentation broth (10 μL) retrieved at the indicated times, lane M: standard protein size marker.

Mentions: To confirm the TFP trap technology using another rarely secreted protein, we screened TFPs for hIL-32, a candidate anticancer and anti-immune disease drug27. The hIL32 gene, flanked with linker DNA and a 0.3-kb DNA fragment encoding an N-terminal part of invertase, was prepared by overlap extension PCR and applied to the TFP library as described before. Around 250 transformants were formed on sucrose medium, compared to 3 × 104 transformants on UD medium. Plasmid DNA was recovered from the 30 largest colonies, and the hIL32 gene was expressed without SUC2 directed by each TFP to select clones that exhibited high levels of hIL-32 protein secretion. Similar to the case of hIL-2, 60% of the TFPs secreted hIL-32 into the culture supernatant with different efficiencies (Fig. 5b). Repeated screening of TFPs for hIL-32 and nucleotide sequencing of 30 high-producing clones revealed 14 different TFPs, including four common TFPs also identified in the screening for hIL-2 (Table 1). Some TFPs were repeatedly found from the same genes, but with different sizes (TFP5 and TFP14). Finally, TFP10 was selected, and fed-batch fermentation was carried out to confirm the secretory productivity of hIL-32. As a result, over 500 mg/L of hIL-32 was produced from 60 g DCW/L of yeast (Fig. 6).


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)

Fermentation of recombinant yeast secreting hIL-32 directed by TFP10.(a) Profile of fed-batch fermentation, (b) SDS-PAGE followed by Coomassie staining analysis of fermentation broth (10 μL) retrieved at the indicated times, lane M: standard protein size marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Fermentation of recombinant yeast secreting hIL-32 directed by TFP10.(a) Profile of fed-batch fermentation, (b) SDS-PAGE followed by Coomassie staining analysis of fermentation broth (10 μL) retrieved at the indicated times, lane M: standard protein size marker.
Mentions: To confirm the TFP trap technology using another rarely secreted protein, we screened TFPs for hIL-32, a candidate anticancer and anti-immune disease drug27. The hIL32 gene, flanked with linker DNA and a 0.3-kb DNA fragment encoding an N-terminal part of invertase, was prepared by overlap extension PCR and applied to the TFP library as described before. Around 250 transformants were formed on sucrose medium, compared to 3 × 104 transformants on UD medium. Plasmid DNA was recovered from the 30 largest colonies, and the hIL32 gene was expressed without SUC2 directed by each TFP to select clones that exhibited high levels of hIL-32 protein secretion. Similar to the case of hIL-2, 60% of the TFPs secreted hIL-32 into the culture supernatant with different efficiencies (Fig. 5b). Repeated screening of TFPs for hIL-32 and nucleotide sequencing of 30 high-producing clones revealed 14 different TFPs, including four common TFPs also identified in the screening for hIL-2 (Table 1). Some TFPs were repeatedly found from the same genes, but with different sizes (TFP5 and TFP14). Finally, TFP10 was selected, and fed-batch fermentation was carried out to confirm the secretory productivity of hIL-32. As a result, over 500 mg/L of hIL-32 was produced from 60 g DCW/L of yeast (Fig. 6).

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