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Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield.

Bonander N, Darby RA, Grgic L, Bora N, Wen J, Brogna S, Poyner DR, O'Neill MA, Bill RM - Microb. Cell Fact. (2009)

Bottom Line: We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets.Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1.This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK. r.m.bill@aston.ac.uk.

ABSTRACT

Background: The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts.

Results: We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis.

Conclusion: This work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved.

No MeSH data available.


Related in: MedlinePlus

Maximum yields of Fps1 are achieved by tuning BMS1 transcript number in a doxycycline-repressible system (yTHC). Fps1 yield in shake flasks is presented for the yTHCBMS1 overexpression strain cultured in 2 × CBS medium with varying amounts of doxycycline (the concentration in μg/mL is shown in parentheses next to each point), thereby varying the copies of BMS1 RNA/cell. The y-axis shows the factor improvement over the control, as for Figure 1. For comparison, data for the wild-type and the three deletion strains presented in Figure 1 (srb5Δ, spt3Δ and gcn5Δ) are also shown. Inset is a typical anti-HA-tag immunoblot showing Fps1 yields for 75 μg total wild-type membrane (lane 1), and 15 μg total yTHCBMS1 membranes extracted from shake-flask cultures grown in the presence of 0 (lane 2), 0.25 (lane 3), 0.5 (lane 4) and 10 μg/mL (lane 5) doxycycline. The markers (M) are 62, 98, 188 kDa ascending from the bottom of the gel upwards.
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Figure 2: Maximum yields of Fps1 are achieved by tuning BMS1 transcript number in a doxycycline-repressible system (yTHC). Fps1 yield in shake flasks is presented for the yTHCBMS1 overexpression strain cultured in 2 × CBS medium with varying amounts of doxycycline (the concentration in μg/mL is shown in parentheses next to each point), thereby varying the copies of BMS1 RNA/cell. The y-axis shows the factor improvement over the control, as for Figure 1. For comparison, data for the wild-type and the three deletion strains presented in Figure 1 (srb5Δ, spt3Δ and gcn5Δ) are also shown. Inset is a typical anti-HA-tag immunoblot showing Fps1 yields for 75 μg total wild-type membrane (lane 1), and 15 μg total yTHCBMS1 membranes extracted from shake-flask cultures grown in the presence of 0 (lane 2), 0.25 (lane 3), 0.5 (lane 4) and 10 μg/mL (lane 5) doxycycline. The markers (M) are 62, 98, 188 kDa ascending from the bottom of the gel upwards.

Mentions: We next used a doxycycline-repressible BMS1 overexpression strain from the Open Biosystems yTHC collection (Thermo Fisher Scientific, UK) that allows gene expression to be tuned by the addition of doxycycline to the growth medium. This permitted further examination of the correlation between up-regulation of BMS1 and recombinant Fps1 yield. We found that in shake flask cultures, yield varied as doxycycline concentration (and hence BMS1 transcript number) was varied, as shown in Figure 2. Maximal Fps1 yields of a factor of 78 over wild-type were obtained at 0.5 μg/mL doxycycline. On culturing this strain in a bioreactor in the presence of 0.5 μg/mL doxycycline, the yield improvement over wild-type rose by up to a factor of 137 (data not shown). At 0.5 μg/mL doxycycline, the BMS1 level was 0.5 copies/cell (standard deviation = 0.1; n = 2) compared with that of 0.1 copies/cell in the wild type strain, then decreased with increasing doxycycline concentration (Figure 2).


Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield.

Bonander N, Darby RA, Grgic L, Bora N, Wen J, Brogna S, Poyner DR, O'Neill MA, Bill RM - Microb. Cell Fact. (2009)

Maximum yields of Fps1 are achieved by tuning BMS1 transcript number in a doxycycline-repressible system (yTHC). Fps1 yield in shake flasks is presented for the yTHCBMS1 overexpression strain cultured in 2 × CBS medium with varying amounts of doxycycline (the concentration in μg/mL is shown in parentheses next to each point), thereby varying the copies of BMS1 RNA/cell. The y-axis shows the factor improvement over the control, as for Figure 1. For comparison, data for the wild-type and the three deletion strains presented in Figure 1 (srb5Δ, spt3Δ and gcn5Δ) are also shown. Inset is a typical anti-HA-tag immunoblot showing Fps1 yields for 75 μg total wild-type membrane (lane 1), and 15 μg total yTHCBMS1 membranes extracted from shake-flask cultures grown in the presence of 0 (lane 2), 0.25 (lane 3), 0.5 (lane 4) and 10 μg/mL (lane 5) doxycycline. The markers (M) are 62, 98, 188 kDa ascending from the bottom of the gel upwards.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654770&req=5

Figure 2: Maximum yields of Fps1 are achieved by tuning BMS1 transcript number in a doxycycline-repressible system (yTHC). Fps1 yield in shake flasks is presented for the yTHCBMS1 overexpression strain cultured in 2 × CBS medium with varying amounts of doxycycline (the concentration in μg/mL is shown in parentheses next to each point), thereby varying the copies of BMS1 RNA/cell. The y-axis shows the factor improvement over the control, as for Figure 1. For comparison, data for the wild-type and the three deletion strains presented in Figure 1 (srb5Δ, spt3Δ and gcn5Δ) are also shown. Inset is a typical anti-HA-tag immunoblot showing Fps1 yields for 75 μg total wild-type membrane (lane 1), and 15 μg total yTHCBMS1 membranes extracted from shake-flask cultures grown in the presence of 0 (lane 2), 0.25 (lane 3), 0.5 (lane 4) and 10 μg/mL (lane 5) doxycycline. The markers (M) are 62, 98, 188 kDa ascending from the bottom of the gel upwards.
Mentions: We next used a doxycycline-repressible BMS1 overexpression strain from the Open Biosystems yTHC collection (Thermo Fisher Scientific, UK) that allows gene expression to be tuned by the addition of doxycycline to the growth medium. This permitted further examination of the correlation between up-regulation of BMS1 and recombinant Fps1 yield. We found that in shake flask cultures, yield varied as doxycycline concentration (and hence BMS1 transcript number) was varied, as shown in Figure 2. Maximal Fps1 yields of a factor of 78 over wild-type were obtained at 0.5 μg/mL doxycycline. On culturing this strain in a bioreactor in the presence of 0.5 μg/mL doxycycline, the yield improvement over wild-type rose by up to a factor of 137 (data not shown). At 0.5 μg/mL doxycycline, the BMS1 level was 0.5 copies/cell (standard deviation = 0.1; n = 2) compared with that of 0.1 copies/cell in the wild type strain, then decreased with increasing doxycycline concentration (Figure 2).

Bottom Line: We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets.Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1.This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK. r.m.bill@aston.ac.uk.

ABSTRACT

Background: The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts.

Results: We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis.

Conclusion: This work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved.

No MeSH data available.


Related in: MedlinePlus