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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

Production and characterization of hIL-2 directed by translational fusion partner (TFP) 1–4 in S. cerevisiae.(a) Profile of fed-batch fermentation. Closed circles: cell growth, closed square: concentration of glucose, closed triangle: concentration of galactose, open diamond: concentration of hIL-2. (b) SDS-PAGE analysis of 10-μL aliquots of fermentation broth retrieved at the indicated times. M: size markers. (c) SDS-PAGE for the purified hIL-2. Lane 1: after ultrafiltration, lane 2: after ion-exchange chromatography, and lane 3: after gel filtration chromatography. (d) Bioactivity assay of the purified hIL-2. The EL-4 cell line was cultured in the presence of indicated amount of hIL-2, and cell proliferation was analysed by bromodeoxyuridine (BrdU) labelling. The protein was revealed by Coomassie staining.
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f3: Production and characterization of hIL-2 directed by translational fusion partner (TFP) 1–4 in S. cerevisiae.(a) Profile of fed-batch fermentation. Closed circles: cell growth, closed square: concentration of glucose, closed triangle: concentration of galactose, open diamond: concentration of hIL-2. (b) SDS-PAGE analysis of 10-μL aliquots of fermentation broth retrieved at the indicated times. M: size markers. (c) SDS-PAGE for the purified hIL-2. Lane 1: after ultrafiltration, lane 2: after ion-exchange chromatography, and lane 3: after gel filtration chromatography. (d) Bioactivity assay of the purified hIL-2. The EL-4 cell line was cultured in the presence of indicated amount of hIL-2, and cell proliferation was analysed by bromodeoxyuridine (BrdU) labelling. The protein was revealed by Coomassie staining.

Mentions: Fed-batch fermentation of the Y2805 strain harbouring YGaTFP1-4-hIL2 was carried out to confirm the productivity of hIL-2. The yield of secreted hIL-2 was over 400 mg/L from 40 g DCW/L (Fig. 3a,b), as determined by a density comparison with standard hIL-2 produced by E. coli. The intactness of the secreted recombinant hIL-2 was confirmed by N-terminal amino acid sequencing and mass analysis (data not shown). To analyse the activity of secreted recombinant hIL-2, the protein was purified by ion-exchange and gel-filtration column chromatography (Fig. 3c). The biological activity of the purified hIL-2 was determined by an in vitro proliferation assay using the EL-4 (mouse T-lymphocyte) cell line (Fig. 3d). Recombinant yeast hIL-2 showed biological activity similar to that of standard hIL-2, suggesting that the hypersecreted yeast hIL-2 produced using the TFP technology developed in this study was fully intact. Furthermore, this screening system could directly retrieve optimal TFPs facilitating the hypersecretion of low-secretion-competent proteins.


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)

Production and characterization of hIL-2 directed by translational fusion partner (TFP) 1–4 in S. cerevisiae.(a) Profile of fed-batch fermentation. Closed circles: cell growth, closed square: concentration of glucose, closed triangle: concentration of galactose, open diamond: concentration of hIL-2. (b) SDS-PAGE analysis of 10-μL aliquots of fermentation broth retrieved at the indicated times. M: size markers. (c) SDS-PAGE for the purified hIL-2. Lane 1: after ultrafiltration, lane 2: after ion-exchange chromatography, and lane 3: after gel filtration chromatography. (d) Bioactivity assay of the purified hIL-2. The EL-4 cell line was cultured in the presence of indicated amount of hIL-2, and cell proliferation was analysed by bromodeoxyuridine (BrdU) labelling. The protein was revealed by Coomassie staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Production and characterization of hIL-2 directed by translational fusion partner (TFP) 1–4 in S. cerevisiae.(a) Profile of fed-batch fermentation. Closed circles: cell growth, closed square: concentration of glucose, closed triangle: concentration of galactose, open diamond: concentration of hIL-2. (b) SDS-PAGE analysis of 10-μL aliquots of fermentation broth retrieved at the indicated times. M: size markers. (c) SDS-PAGE for the purified hIL-2. Lane 1: after ultrafiltration, lane 2: after ion-exchange chromatography, and lane 3: after gel filtration chromatography. (d) Bioactivity assay of the purified hIL-2. The EL-4 cell line was cultured in the presence of indicated amount of hIL-2, and cell proliferation was analysed by bromodeoxyuridine (BrdU) labelling. The protein was revealed by Coomassie staining.
Mentions: Fed-batch fermentation of the Y2805 strain harbouring YGaTFP1-4-hIL2 was carried out to confirm the productivity of hIL-2. The yield of secreted hIL-2 was over 400 mg/L from 40 g DCW/L (Fig. 3a,b), as determined by a density comparison with standard hIL-2 produced by E. coli. The intactness of the secreted recombinant hIL-2 was confirmed by N-terminal amino acid sequencing and mass analysis (data not shown). To analyse the activity of secreted recombinant hIL-2, the protein was purified by ion-exchange and gel-filtration column chromatography (Fig. 3c). The biological activity of the purified hIL-2 was determined by an in vitro proliferation assay using the EL-4 (mouse T-lymphocyte) cell line (Fig. 3d). Recombinant yeast hIL-2 showed biological activity similar to that of standard hIL-2, suggesting that the hypersecreted yeast hIL-2 produced using the TFP technology developed in this study was fully intact. Furthermore, this screening system could directly retrieve optimal TFPs facilitating the hypersecretion of low-secretion-competent proteins.

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