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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

SDS-PAGE analysis of hIL-2 (a) and hIL-32 (b) expressed by randomly selected translational fusion partners from TFP library.A 0.6-mL aliquot of culture supernatant was precipitated with acetone and analysed on a 10% Tricine gel. M: standard protein size marker, C: host strain carrying a mock vector, (a) Lane 1: TFP1-4, lane 2, 9, 13: TFP5, lane 3: TFP18, lane 5, 22: TFP19-1, lane 10: TFP16-1, lane 11, 15, 18: TFP17-1, lane 16: TFP5-3, lane 17, 19, 20, 21: TFP17-3. (b) Lane 1, 14, 22: TFP10, lane 6, 11, 21, 23: TFP5-1, lane 7: TFP6, lane 12: TFP7-1, lane 16: TFP18-1, lane 17: TFP20, lane 18, 25: TFP5-2, lane 19: TFP11, lane 20: TFP14-1, lane 26: TFP21, lane 27: TFP16-3, lane 28: TFP21, lane 30: TFP22. The protein was revealed by Coomassie staining.
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f5: SDS-PAGE analysis of hIL-2 (a) and hIL-32 (b) expressed by randomly selected translational fusion partners from TFP library.A 0.6-mL aliquot of culture supernatant was precipitated with acetone and analysed on a 10% Tricine gel. M: standard protein size marker, C: host strain carrying a mock vector, (a) Lane 1: TFP1-4, lane 2, 9, 13: TFP5, lane 3: TFP18, lane 5, 22: TFP19-1, lane 10: TFP16-1, lane 11, 15, 18: TFP17-1, lane 16: TFP5-3, lane 17, 19, 20, 21: TFP17-3. (b) Lane 1, 14, 22: TFP10, lane 6, 11, 21, 23: TFP5-1, lane 7: TFP6, lane 12: TFP7-1, lane 16: TFP18-1, lane 17: TFP20, lane 18, 25: TFP5-2, lane 19: TFP11, lane 20: TFP14-1, lane 26: TFP21, lane 27: TFP16-3, lane 28: TFP21, lane 30: TFP22. The protein was revealed by Coomassie staining.

Mentions: To confirm actual hIL-2 secretion, plasmids were recovered from 24 transformants, and hIL-2 expression vectors were reconstructed to express hIL2 without SUC2 directed by respective TFPs. Around 60% of TFPs secreted hIL-2 into culture supernatants with variable efficiencies (Fig. 5a). Several TFPs showed efficiencies comparable to that of TFP1–4, but some TFPs secreted only small amounts of hIL-2, which were detectable by western blotting with an anti-hIL-2 antibody (data not shown). Nucleotide sequencing of TFPs secreting hIL-2 revealed seven different TFPs. To obtain additional TFPs, we repeated this process two times more and finally obtained 28 different TFPs, as summarised in Table 1. All TFPs originated from various types of secretory proteins, such as cell wall proteins, plasma and vacuolar membrane proteins, and functionally unknown secretory proteins. Some proteins were repeatedly found with different sizes from the same genes (TFP3, −5, −11, −16, −17, and −19). Indeed, most of the yeast proteins passing through the ER could be TFP candidates. Consequently, there were numerous unidentified TFP candidates in the yeast genome that could improve the secretion competency of hIL-2. Among them, TFP1–4 seemed to be an optimal TFP for secretion of hIL-2.


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)

SDS-PAGE analysis of hIL-2 (a) and hIL-32 (b) expressed by randomly selected translational fusion partners from TFP library.A 0.6-mL aliquot of culture supernatant was precipitated with acetone and analysed on a 10% Tricine gel. M: standard protein size marker, C: host strain carrying a mock vector, (a) Lane 1: TFP1-4, lane 2, 9, 13: TFP5, lane 3: TFP18, lane 5, 22: TFP19-1, lane 10: TFP16-1, lane 11, 15, 18: TFP17-1, lane 16: TFP5-3, lane 17, 19, 20, 21: TFP17-3. (b) Lane 1, 14, 22: TFP10, lane 6, 11, 21, 23: TFP5-1, lane 7: TFP6, lane 12: TFP7-1, lane 16: TFP18-1, lane 17: TFP20, lane 18, 25: TFP5-2, lane 19: TFP11, lane 20: TFP14-1, lane 26: TFP21, lane 27: TFP16-3, lane 28: TFP21, lane 30: TFP22. 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

f5: SDS-PAGE analysis of hIL-2 (a) and hIL-32 (b) expressed by randomly selected translational fusion partners from TFP library.A 0.6-mL aliquot of culture supernatant was precipitated with acetone and analysed on a 10% Tricine gel. M: standard protein size marker, C: host strain carrying a mock vector, (a) Lane 1: TFP1-4, lane 2, 9, 13: TFP5, lane 3: TFP18, lane 5, 22: TFP19-1, lane 10: TFP16-1, lane 11, 15, 18: TFP17-1, lane 16: TFP5-3, lane 17, 19, 20, 21: TFP17-3. (b) Lane 1, 14, 22: TFP10, lane 6, 11, 21, 23: TFP5-1, lane 7: TFP6, lane 12: TFP7-1, lane 16: TFP18-1, lane 17: TFP20, lane 18, 25: TFP5-2, lane 19: TFP11, lane 20: TFP14-1, lane 26: TFP21, lane 27: TFP16-3, lane 28: TFP21, lane 30: TFP22. The protein was revealed by Coomassie staining.
Mentions: To confirm actual hIL-2 secretion, plasmids were recovered from 24 transformants, and hIL-2 expression vectors were reconstructed to express hIL2 without SUC2 directed by respective TFPs. Around 60% of TFPs secreted hIL-2 into culture supernatants with variable efficiencies (Fig. 5a). Several TFPs showed efficiencies comparable to that of TFP1–4, but some TFPs secreted only small amounts of hIL-2, which were detectable by western blotting with an anti-hIL-2 antibody (data not shown). Nucleotide sequencing of TFPs secreting hIL-2 revealed seven different TFPs. To obtain additional TFPs, we repeated this process two times more and finally obtained 28 different TFPs, as summarised in Table 1. All TFPs originated from various types of secretory proteins, such as cell wall proteins, plasma and vacuolar membrane proteins, and functionally unknown secretory proteins. Some proteins were repeatedly found with different sizes from the same genes (TFP3, −5, −11, −16, −17, and −19). Indeed, most of the yeast proteins passing through the ER could be TFP candidates. Consequently, there were numerous unidentified TFP candidates in the yeast genome that could improve the secretion competency of hIL-2. Among them, TFP1–4 seemed to be an optimal TFP for secretion of hIL-2.

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