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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

The yTHCBMS1 strain, which produces the highest yields of Fps1, can also be used to improve the functional yield of other proteins. (A) Binding of ZM241385 on yeast membranes producing the G-protein coupled receptor, hA2aR is shown for duplicate determinations. Error bars represent the standard deviation (n = 2). Also shown are the Kd (nM) and Bmax (pmol receptor/mg protein) values with standard deviation given in parentheses (n = 2). (B) The yTHCBMS1 strain (grey bars) improves the functional yield of green fluorescent protein by a factor of 2 over wild-type (white bars). Error bars represent the standard deviation (n = 3). Shown is the total GFP yield from a 50 mL culture of OD600 1.0.
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Figure 3: The yTHCBMS1 strain, which produces the highest yields of Fps1, can also be used to improve the functional yield of other proteins. (A) Binding of ZM241385 on yeast membranes producing the G-protein coupled receptor, hA2aR is shown for duplicate determinations. Error bars represent the standard deviation (n = 2). Also shown are the Kd (nM) and Bmax (pmol receptor/mg protein) values with standard deviation given in parentheses (n = 2). (B) The yTHCBMS1 strain (grey bars) improves the functional yield of green fluorescent protein by a factor of 2 over wild-type (white bars). Error bars represent the standard deviation (n = 3). Shown is the total GFP yield from a 50 mL culture of OD600 1.0.

Mentions: In order to examine whether our screening strategy using Fps1 as a target, had resulted in an Fps1-specific result, we tested the human G-protein coupled receptor, A2aR in our best-performing strain. Figure 3A shows that the improvement in active hA2aR as assessed by radioligand binding was small for cells cultured under conditions optimized for Fps1 production (0.5 μg/mL doxycycline). On examining whether hA2aR production could be tuned further, we confirmed that maximal binding activity was obtained at a doxycycline concentration of 10 μg/mL (where BMS1 levels were approximately three times those in wild-type cells). Analysis of the binding curves confirmed that this was due to an increase in receptor expression (i.e. the maximum amount of ZM241385 bound) and not because of any change in the affinity of the receptor for the radioligand. Furthermore, the strain could be tuned to maximize yields of functional green fluorescent protein, doubling wild-type yields at 10 μg/mL doxycycline when grown in 50 mL shake-flask cultures (Figure 3B). These results suggested that the up-regulation of BMS1 in a target-protein-specific manner might be the key to maximizing yields for a range of proteins.


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)

The yTHCBMS1 strain, which produces the highest yields of Fps1, can also be used to improve the functional yield of other proteins. (A) Binding of ZM241385 on yeast membranes producing the G-protein coupled receptor, hA2aR is shown for duplicate determinations. Error bars represent the standard deviation (n = 2). Also shown are the Kd (nM) and Bmax (pmol receptor/mg protein) values with standard deviation given in parentheses (n = 2). (B) The yTHCBMS1 strain (grey bars) improves the functional yield of green fluorescent protein by a factor of 2 over wild-type (white bars). Error bars represent the standard deviation (n = 3). Shown is the total GFP yield from a 50 mL culture of OD600 1.0.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: The yTHCBMS1 strain, which produces the highest yields of Fps1, can also be used to improve the functional yield of other proteins. (A) Binding of ZM241385 on yeast membranes producing the G-protein coupled receptor, hA2aR is shown for duplicate determinations. Error bars represent the standard deviation (n = 2). Also shown are the Kd (nM) and Bmax (pmol receptor/mg protein) values with standard deviation given in parentheses (n = 2). (B) The yTHCBMS1 strain (grey bars) improves the functional yield of green fluorescent protein by a factor of 2 over wild-type (white bars). Error bars represent the standard deviation (n = 3). Shown is the total GFP yield from a 50 mL culture of OD600 1.0.
Mentions: In order to examine whether our screening strategy using Fps1 as a target, had resulted in an Fps1-specific result, we tested the human G-protein coupled receptor, A2aR in our best-performing strain. Figure 3A shows that the improvement in active hA2aR as assessed by radioligand binding was small for cells cultured under conditions optimized for Fps1 production (0.5 μg/mL doxycycline). On examining whether hA2aR production could be tuned further, we confirmed that maximal binding activity was obtained at a doxycycline concentration of 10 μg/mL (where BMS1 levels were approximately three times those in wild-type cells). Analysis of the binding curves confirmed that this was due to an increase in receptor expression (i.e. the maximum amount of ZM241385 bound) and not because of any change in the affinity of the receptor for the radioligand. Furthermore, the strain could be tuned to maximize yields of functional green fluorescent protein, doubling wild-type yields at 10 μg/mL doxycycline when grown in 50 mL shake-flask cultures (Figure 3B). These results suggested that the up-regulation of BMS1 in a target-protein-specific manner might be the key to maximizing yields for a range of proteins.

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