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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

Effects of the translational fusion partner (TFP) 1 domains on the secretion of a rarely secreted protein, human interleukin (hIL)-2.(a) Schematic diagram of TFP1 derivatives. TFP1 was divided into five domains (UTR: 5′ untranslated region, A: signal sequence, B: N-glycosylation site, C: Ser and Ala rich sequence, D: a flanking sequence). Deleted domains indicated by the bent line. (b) SDS-PAGE analysis of hIL-2 secreted into the culture medium by recombinant strains carrying the plasmids harbouring TFP1 derivatives. Lane S: recombinant hIL-2 produced by Escherichia coli. M: standard protein size marker. The protein is revealed by Coomassie staining. (c) Western blot analysis of intracellular and extracellular proteins produced by recombinant strains carrying the indicated plasmids. C: host strain carrying the mock vector. (d) SDS-PAGE followed by Coomassie staining for the comparison of hIL-2 secretion using different signal sequences. Lane 1: α-amylase signal peptide from B. subtilis, lane 2: prepro signal peptide of mating factor α from S. cerevisiae, and lane 3: TFP1-4 in this study.
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f2: Effects of the translational fusion partner (TFP) 1 domains on the secretion of a rarely secreted protein, human interleukin (hIL)-2.(a) Schematic diagram of TFP1 derivatives. TFP1 was divided into five domains (UTR: 5′ untranslated region, A: signal sequence, B: N-glycosylation site, C: Ser and Ala rich sequence, D: a flanking sequence). Deleted domains indicated by the bent line. (b) SDS-PAGE analysis of hIL-2 secreted into the culture medium by recombinant strains carrying the plasmids harbouring TFP1 derivatives. Lane S: recombinant hIL-2 produced by Escherichia coli. M: standard protein size marker. The protein is revealed by Coomassie staining. (c) Western blot analysis of intracellular and extracellular proteins produced by recombinant strains carrying the indicated plasmids. C: host strain carrying the mock vector. (d) SDS-PAGE followed by Coomassie staining for the comparison of hIL-2 secretion using different signal sequences. Lane 1: α-amylase signal peptide from B. subtilis, lane 2: prepro signal peptide of mating factor α from S. cerevisiae, and lane 3: TFP1-4 in this study.

Mentions: To ensure secretion of hIL-2 using the screened TFPs without invertase, the SUC2 gene of each vector was removed, and the yeast dipeptidyl protease Kex2p25 cleavage site (LDKR) was inserted between TFP and hIL2 by PCR. Among three tested TFPs, TFP1 was superior to the other TFPs for hIL-2 secretion (data not shown). The TFP1 nucleotide sequence contained five domains that might be important for secretion of hIL-2 (domain A: signal sequence, B: N-glycosylation site, C: Ser- and Ala-rich sequence, D: a flanking sequence, and a 5′-untranslated region [UTR]). Four truncated TFP1 derivatives were constructed to identify the effects of each domain on hIL-2 secretion (Fig. 2a), and the culture supernatants of each construct were analysed by SDS-PAGE (Fig. 2b). The hIL-2 protein fused with TFP1-1 and TFP1-2, containing the signal sequence or N-glycosylation site only, was not secreted or even detected by western blot analysis, but was detected in the intracellular fraction of each cell (Fig. 2c). The slightly larger size of these proteins compared with the secreted hIL-2 indicated that they were not processed by Kex2p. Therefore, these fusion proteins may not reach the Golgi complex and would be caught by the ER quality control system. This result clearly showed that hIL-2 was not secreted by a traditional approach employing only a secretion signal. TFP1-3, containing domains A, B, and C, secreted authentic hIL-2 with a small amount of unprocessed and degraded protein, but still less than that of the original TFP1 (Fig. 2b), suggesting that all TFP1 domains were necessary for hIL-2 secretion. Finally, hIL-2 secretion was augmented by about three-fold after removing the 5′-UTR (TFP1–4) (Fig. 2b). Some yeast genes are known to contain negative cis-acting elements that affect the translational efficiency on the 5′-UTR26. The secretion-enhancing effects of TFP1-4 were compared with other well-known signal sequences often used for secretion of recombinant proteins in yeast. Compared with the α-amylase (AMY) signal peptide from Bacillus subtilis and mating factor α signal peptide (MFα) of S. cerevisiae, TFP1-4 dramatically improved hIL2 secretory productivity by about 10-fold compared with that of MFα and several hundred-fold compared with that of AMY, respectively (Fig. 2d).


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)

Effects of the translational fusion partner (TFP) 1 domains on the secretion of a rarely secreted protein, human interleukin (hIL)-2.(a) Schematic diagram of TFP1 derivatives. TFP1 was divided into five domains (UTR: 5′ untranslated region, A: signal sequence, B: N-glycosylation site, C: Ser and Ala rich sequence, D: a flanking sequence). Deleted domains indicated by the bent line. (b) SDS-PAGE analysis of hIL-2 secreted into the culture medium by recombinant strains carrying the plasmids harbouring TFP1 derivatives. Lane S: recombinant hIL-2 produced by Escherichia coli. M: standard protein size marker. The protein is revealed by Coomassie staining. (c) Western blot analysis of intracellular and extracellular proteins produced by recombinant strains carrying the indicated plasmids. C: host strain carrying the mock vector. (d) SDS-PAGE followed by Coomassie staining for the comparison of hIL-2 secretion using different signal sequences. Lane 1: α-amylase signal peptide from B. subtilis, lane 2: prepro signal peptide of mating factor α from S. cerevisiae, and lane 3: TFP1-4 in this study.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Effects of the translational fusion partner (TFP) 1 domains on the secretion of a rarely secreted protein, human interleukin (hIL)-2.(a) Schematic diagram of TFP1 derivatives. TFP1 was divided into five domains (UTR: 5′ untranslated region, A: signal sequence, B: N-glycosylation site, C: Ser and Ala rich sequence, D: a flanking sequence). Deleted domains indicated by the bent line. (b) SDS-PAGE analysis of hIL-2 secreted into the culture medium by recombinant strains carrying the plasmids harbouring TFP1 derivatives. Lane S: recombinant hIL-2 produced by Escherichia coli. M: standard protein size marker. The protein is revealed by Coomassie staining. (c) Western blot analysis of intracellular and extracellular proteins produced by recombinant strains carrying the indicated plasmids. C: host strain carrying the mock vector. (d) SDS-PAGE followed by Coomassie staining for the comparison of hIL-2 secretion using different signal sequences. Lane 1: α-amylase signal peptide from B. subtilis, lane 2: prepro signal peptide of mating factor α from S. cerevisiae, and lane 3: TFP1-4 in this study.
Mentions: To ensure secretion of hIL-2 using the screened TFPs without invertase, the SUC2 gene of each vector was removed, and the yeast dipeptidyl protease Kex2p25 cleavage site (LDKR) was inserted between TFP and hIL2 by PCR. Among three tested TFPs, TFP1 was superior to the other TFPs for hIL-2 secretion (data not shown). The TFP1 nucleotide sequence contained five domains that might be important for secretion of hIL-2 (domain A: signal sequence, B: N-glycosylation site, C: Ser- and Ala-rich sequence, D: a flanking sequence, and a 5′-untranslated region [UTR]). Four truncated TFP1 derivatives were constructed to identify the effects of each domain on hIL-2 secretion (Fig. 2a), and the culture supernatants of each construct were analysed by SDS-PAGE (Fig. 2b). The hIL-2 protein fused with TFP1-1 and TFP1-2, containing the signal sequence or N-glycosylation site only, was not secreted or even detected by western blot analysis, but was detected in the intracellular fraction of each cell (Fig. 2c). The slightly larger size of these proteins compared with the secreted hIL-2 indicated that they were not processed by Kex2p. Therefore, these fusion proteins may not reach the Golgi complex and would be caught by the ER quality control system. This result clearly showed that hIL-2 was not secreted by a traditional approach employing only a secretion signal. TFP1-3, containing domains A, B, and C, secreted authentic hIL-2 with a small amount of unprocessed and degraded protein, but still less than that of the original TFP1 (Fig. 2b), suggesting that all TFP1 domains were necessary for hIL-2 secretion. Finally, hIL-2 secretion was augmented by about three-fold after removing the 5′-UTR (TFP1–4) (Fig. 2b). Some yeast genes are known to contain negative cis-acting elements that affect the translational efficiency on the 5′-UTR26. The secretion-enhancing effects of TFP1-4 were compared with other well-known signal sequences often used for secretion of recombinant proteins in yeast. Compared with the α-amylase (AMY) signal peptide from Bacillus subtilis and mating factor α signal peptide (MFα) of S. cerevisiae, TFP1-4 dramatically improved hIL2 secretory productivity by about 10-fold compared with that of MFα and several hundred-fold compared with that of AMY, respectively (Fig. 2d).

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