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

Application of the mini-translational fusion partner (TFP) library for the production of hGH.(a) Schematic diagram of direct TFP screening from a mini TFP library. (b) SDS-PAGE analysis of hGH expressed by selected translational fusion partners (TFPs). A 0.6-mL aliquot of the culture supernatant was analysed on a 10% Tricine gel after precipitation with acetone. M: standard protein size marker, C: host strain carrying a mock vector, lane 2, 5, 6: TFP5-1, lane 9: TFP4, lane 10, 13: TFP11, lane 11, 27: TFP17-3. (c) SDS-PAGE analysis of fed-batch fermentation broth (10 μL) retrieved at the indicated times. (d) Bioactivity assay of the purified hGH. The Nb2-lymphoma cell line was cultured in the presence of the indicated amounts of hGH, and cell proliferation was analysed at 490 nm. The protein was revealed by Coomassie staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Application of the mini-translational fusion partner (TFP) library for the production of hGH.(a) Schematic diagram of direct TFP screening from a mini TFP library. (b) SDS-PAGE analysis of hGH expressed by selected translational fusion partners (TFPs). A 0.6-mL aliquot of the culture supernatant was analysed on a 10% Tricine gel after precipitation with acetone. M: standard protein size marker, C: host strain carrying a mock vector, lane 2, 5, 6: TFP5-1, lane 9: TFP4, lane 10, 13: TFP11, lane 11, 27: TFP17-3. (c) SDS-PAGE analysis of fed-batch fermentation broth (10 μL) retrieved at the indicated times. (d) Bioactivity assay of the purified hGH. The Nb2-lymphoma cell line was cultured in the presence of the indicated amounts of hGH, and cell proliferation was analysed at 490 nm. The protein was revealed by Coomassie staining.

Mentions: As summarised in Table 1, the 39 TFPs selected by two rarely secreted target proteins were derived from various secretory proteins with different functions and localisations. The lengths of TFPs varied from 51 to 213 aa. Most TFPs were parts of extracellular glycoproteins or membrane proteins with apparent secretion signal sequences. Among them, TFP1-4 and TFP10 were selected as optimal TFPs for the secretion of hIL-2 and hIL-32, respectively. Although the remaining TFPs showed less capacity to induce the secretion of hIL-2 and hIL-32 than the selected TFPs, they were tested for the secretory production of other proteins to demonstrate our initial concept of the different capacities of TFP according to the target proteins. Mini-library vectors containing 39 TFPs were used for the selection of an optimal TFP for the secretion of human growth hormone (hGH). Vectors containing each TFP were constructed as described in the Methods for the simple insertion of target genes between TFPs and the transcriptional terminator. Y2805 was transformed with a mixture of SwaI-digested mini-library vectors and the PCR-amplified hGH gene flanked with homologous sequences of vector ends to construct a mini-library of transformants through in vivo recombination (Fig. 7a). Thirty transformants were selected and cultured to isolate an optimal TFP for the secretion of hGH (Fig. 7b). Most transformants secreted correctly processed hGH (21 kDa) through Kex2p, but with different secretion levels. Yeast strains secreting hGH directed by TFP5-1 (SRL1-59aa), TFP11 (ECM33-55aa), and TFP17-3 (CCW12-125aa) showed higher levels of hGH secretion than others. Although these TFPs were initially isolated for hIL-2 or hIL-32 secretion, they were found to be much more useful for hGH secretion, demonstrating the versatile capabilities of the selected TFPs with regard to the target proteins and the usefulness of the mini-TFP library for the expression of other recombinant proteins. The productivity of hGH was confirmed by fed-batch fermentation of the transformant secreting hGH directed by of TFP5-1. Over 1 g/L of recombinant hGH was secreted from 54 g yeast DCW/L (Fig. 7c). The bioactivity of purified hGH was equivalent to that of standard hGH (Fig. 7d). Another example of TFP selection from the mini-TFP library was performed using an industrial enzyme, pectin-degrading endopolygalacturonase of yeast Kluyveromyces marxianus (KmEPG1). A set of 24 TFPs selected from Table 1 were tested individually for the secretion of KmEPG1. Although most of the selected TFPs rendered efficient secretion of KmEPG1 (Fig. 8a), analysis of EPG activity revealed that transformants, including vectors with TFP1-4 (YAR066W-105aa), TFP3 (CIS3-103aa), and TFP16-3 (SCW4-111aa), were higher than others. Selected TFPs for the optimal secretion of KmEPG were quite different from those for hGH, suggesting the different secretion effects of the TFPs on different target proteins. Around 1 g/L of KmEPG1 was secreted from 45 g DCW/L during fed-batch fermentation of a yeast transformant secreting KmEPG1 directed by TFP3 (Fig. 8b). Although KmEPG1 was reported as a poorly secreted protein in recombinant expression using Pichia pastoris by the MFα signal sequence of S. cerevisiae28 and even under its own secretion signal29, approximately 10-fold more KmEPG1 secretion was detected using TFP3 as a leader sequence instead of its own secretion signal (Fig. 8c). The results clearly suggested that screening for optimal TFPs could improve the secretion level of each protein and that the TFP selection system developed in this study can be a powerful tool for improving the yields of various recombinant 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)

Application of the mini-translational fusion partner (TFP) library for the production of hGH.(a) Schematic diagram of direct TFP screening from a mini TFP library. (b) SDS-PAGE analysis of hGH expressed by selected translational fusion partners (TFPs). A 0.6-mL aliquot of the culture supernatant was analysed on a 10% Tricine gel after precipitation with acetone. M: standard protein size marker, C: host strain carrying a mock vector, lane 2, 5, 6: TFP5-1, lane 9: TFP4, lane 10, 13: TFP11, lane 11, 27: TFP17-3. (c) SDS-PAGE analysis of fed-batch fermentation broth (10 μL) retrieved at the indicated times. (d) Bioactivity assay of the purified hGH. The Nb2-lymphoma cell line was cultured in the presence of the indicated amounts of hGH, and cell proliferation was analysed at 490 nm. 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

f7: Application of the mini-translational fusion partner (TFP) library for the production of hGH.(a) Schematic diagram of direct TFP screening from a mini TFP library. (b) SDS-PAGE analysis of hGH expressed by selected translational fusion partners (TFPs). A 0.6-mL aliquot of the culture supernatant was analysed on a 10% Tricine gel after precipitation with acetone. M: standard protein size marker, C: host strain carrying a mock vector, lane 2, 5, 6: TFP5-1, lane 9: TFP4, lane 10, 13: TFP11, lane 11, 27: TFP17-3. (c) SDS-PAGE analysis of fed-batch fermentation broth (10 μL) retrieved at the indicated times. (d) Bioactivity assay of the purified hGH. The Nb2-lymphoma cell line was cultured in the presence of the indicated amounts of hGH, and cell proliferation was analysed at 490 nm. The protein was revealed by Coomassie staining.
Mentions: As summarised in Table 1, the 39 TFPs selected by two rarely secreted target proteins were derived from various secretory proteins with different functions and localisations. The lengths of TFPs varied from 51 to 213 aa. Most TFPs were parts of extracellular glycoproteins or membrane proteins with apparent secretion signal sequences. Among them, TFP1-4 and TFP10 were selected as optimal TFPs for the secretion of hIL-2 and hIL-32, respectively. Although the remaining TFPs showed less capacity to induce the secretion of hIL-2 and hIL-32 than the selected TFPs, they were tested for the secretory production of other proteins to demonstrate our initial concept of the different capacities of TFP according to the target proteins. Mini-library vectors containing 39 TFPs were used for the selection of an optimal TFP for the secretion of human growth hormone (hGH). Vectors containing each TFP were constructed as described in the Methods for the simple insertion of target genes between TFPs and the transcriptional terminator. Y2805 was transformed with a mixture of SwaI-digested mini-library vectors and the PCR-amplified hGH gene flanked with homologous sequences of vector ends to construct a mini-library of transformants through in vivo recombination (Fig. 7a). Thirty transformants were selected and cultured to isolate an optimal TFP for the secretion of hGH (Fig. 7b). Most transformants secreted correctly processed hGH (21 kDa) through Kex2p, but with different secretion levels. Yeast strains secreting hGH directed by TFP5-1 (SRL1-59aa), TFP11 (ECM33-55aa), and TFP17-3 (CCW12-125aa) showed higher levels of hGH secretion than others. Although these TFPs were initially isolated for hIL-2 or hIL-32 secretion, they were found to be much more useful for hGH secretion, demonstrating the versatile capabilities of the selected TFPs with regard to the target proteins and the usefulness of the mini-TFP library for the expression of other recombinant proteins. The productivity of hGH was confirmed by fed-batch fermentation of the transformant secreting hGH directed by of TFP5-1. Over 1 g/L of recombinant hGH was secreted from 54 g yeast DCW/L (Fig. 7c). The bioactivity of purified hGH was equivalent to that of standard hGH (Fig. 7d). Another example of TFP selection from the mini-TFP library was performed using an industrial enzyme, pectin-degrading endopolygalacturonase of yeast Kluyveromyces marxianus (KmEPG1). A set of 24 TFPs selected from Table 1 were tested individually for the secretion of KmEPG1. Although most of the selected TFPs rendered efficient secretion of KmEPG1 (Fig. 8a), analysis of EPG activity revealed that transformants, including vectors with TFP1-4 (YAR066W-105aa), TFP3 (CIS3-103aa), and TFP16-3 (SCW4-111aa), were higher than others. Selected TFPs for the optimal secretion of KmEPG were quite different from those for hGH, suggesting the different secretion effects of the TFPs on different target proteins. Around 1 g/L of KmEPG1 was secreted from 45 g DCW/L during fed-batch fermentation of a yeast transformant secreting KmEPG1 directed by TFP3 (Fig. 8b). Although KmEPG1 was reported as a poorly secreted protein in recombinant expression using Pichia pastoris by the MFα signal sequence of S. cerevisiae28 and even under its own secretion signal29, approximately 10-fold more KmEPG1 secretion was detected using TFP3 as a leader sequence instead of its own secretion signal (Fig. 8c). The results clearly suggested that screening for optimal TFPs could improve the secretion level of each protein and that the TFP selection system developed in this study can be a powerful tool for improving the yields of various recombinant 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