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Content of intrinsic disorder influences the outcome of cell-free protein synthesis.

Tokmakov AA, Kurotani A, Ikeda M, Terazawa Y, Shirouzu M, Stefanov V, Sakurai T, Yokoyama S - Sci Rep (2015)

Bottom Line: On the one hand, elevated disorder content was associated with the increased ratio of soluble expression.On the other hand, overall propensity for detectable protein expression decreased with disorder content.Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Environmental Genomics, Kobe University, Nada 657-8501, Japan.

ABSTRACT
Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yields with the contents of intrinsic disorder bioinformatically predicted in the expressed sequences. This analysis revealed strong relationships between intrinsic disorder and protein amenability to heterologous cell-free expression. On the one hand, elevated disorder content was associated with the increased ratio of soluble expression. On the other hand, overall propensity for detectable protein expression decreased with disorder content. We further demonstrated that these tendencies are rooted in some distinct features of intrinsically disordered regions, such as low hydrophobicity, elevated surface accessibility and high abundance of sequence motifs for proteolytic degradation, including sites of ubiquitination and PEST sequences. Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

No MeSH data available.


Related in: MedlinePlus

Correlations of cell-free protein expression with contents of intrinsic disorder.Three hundred twenty three (323) non-redundant human amino acid sequences were expressed in an insect cell-free system of protein synthesis under the uniform set of conditions and detected by Western blot analysis. A representative blot is shown in panel (A). The sequences were classified into the three expression categories – soluble (S), insoluble (I) and non-expressed (N). Distribution of the dataset proteins according to disorder contents is presented in panel (B). Positive correlation of soluble protein expression with disorder degree (C) and negative correlation of total detectable protein expression with disorder degree (D) were observed in the expression dataset. Pearson’s pairwise correlation coefficients and their statistical significance are indicated in panels (C,D).
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f1: Correlations of cell-free protein expression with contents of intrinsic disorder.Three hundred twenty three (323) non-redundant human amino acid sequences were expressed in an insect cell-free system of protein synthesis under the uniform set of conditions and detected by Western blot analysis. A representative blot is shown in panel (A). The sequences were classified into the three expression categories – soluble (S), insoluble (I) and non-expressed (N). Distribution of the dataset proteins according to disorder contents is presented in panel (B). Positive correlation of soluble protein expression with disorder degree (C) and negative correlation of total detectable protein expression with disorder degree (D) were observed in the expression dataset. Pearson’s pairwise correlation coefficients and their statistical significance are indicated in panels (C,D).

Mentions: A complete dataset of human proteins and their domains analyzed in this study comprised 323 non-redundant (at 90% level of identity and coverage) amino acid sequences. All proteins in the dataset were synthesized cell-free under the same uniform set of conditions and detected with anti-His antibody, as described in “Methods”. A representative Western blot is shown in Fig. 1A. After evaluation of protein expression levels, each sequence of the dataset was classified into one of the three expression categories – soluble, insoluble or non-expressed, in accordance with the preferential pattern of its expression. The category of non-expressed sequences included the proteins expressed below the detection threshold as well as the proteins expressed at a lower molecular weight than expected. Global estimation of cell-free protein synthesis showed that soluble-expressed proteins represented 34.4%, insoluble-expressed proteins – 16.7%, and non-expressed proteins – 48.9%. Notably, the rate of soluble expression was higher and the rate of insoluble expression was lower than those reported for a related set of human proteins expressed in a bacterial cell-free expression system (25.7% and 46.7%, respectively2). In addition, the rate of non-expressed proteins (48.9%) was higher in the eukaryotic expression system than that observed in the bacterial system (27.6%). These results indicate that, in the investigated systems of heterologous cell-free protein expression, protein synthesis earns better solubility but lower production yield in the eukaryotic system, as compared to those in the bacterial system.


Content of intrinsic disorder influences the outcome of cell-free protein synthesis.

Tokmakov AA, Kurotani A, Ikeda M, Terazawa Y, Shirouzu M, Stefanov V, Sakurai T, Yokoyama S - Sci Rep (2015)

Correlations of cell-free protein expression with contents of intrinsic disorder.Three hundred twenty three (323) non-redundant human amino acid sequences were expressed in an insect cell-free system of protein synthesis under the uniform set of conditions and detected by Western blot analysis. A representative blot is shown in panel (A). The sequences were classified into the three expression categories – soluble (S), insoluble (I) and non-expressed (N). Distribution of the dataset proteins according to disorder contents is presented in panel (B). Positive correlation of soluble protein expression with disorder degree (C) and negative correlation of total detectable protein expression with disorder degree (D) were observed in the expression dataset. Pearson’s pairwise correlation coefficients and their statistical significance are indicated in panels (C,D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4566126&req=5

f1: Correlations of cell-free protein expression with contents of intrinsic disorder.Three hundred twenty three (323) non-redundant human amino acid sequences were expressed in an insect cell-free system of protein synthesis under the uniform set of conditions and detected by Western blot analysis. A representative blot is shown in panel (A). The sequences were classified into the three expression categories – soluble (S), insoluble (I) and non-expressed (N). Distribution of the dataset proteins according to disorder contents is presented in panel (B). Positive correlation of soluble protein expression with disorder degree (C) and negative correlation of total detectable protein expression with disorder degree (D) were observed in the expression dataset. Pearson’s pairwise correlation coefficients and their statistical significance are indicated in panels (C,D).
Mentions: A complete dataset of human proteins and their domains analyzed in this study comprised 323 non-redundant (at 90% level of identity and coverage) amino acid sequences. All proteins in the dataset were synthesized cell-free under the same uniform set of conditions and detected with anti-His antibody, as described in “Methods”. A representative Western blot is shown in Fig. 1A. After evaluation of protein expression levels, each sequence of the dataset was classified into one of the three expression categories – soluble, insoluble or non-expressed, in accordance with the preferential pattern of its expression. The category of non-expressed sequences included the proteins expressed below the detection threshold as well as the proteins expressed at a lower molecular weight than expected. Global estimation of cell-free protein synthesis showed that soluble-expressed proteins represented 34.4%, insoluble-expressed proteins – 16.7%, and non-expressed proteins – 48.9%. Notably, the rate of soluble expression was higher and the rate of insoluble expression was lower than those reported for a related set of human proteins expressed in a bacterial cell-free expression system (25.7% and 46.7%, respectively2). In addition, the rate of non-expressed proteins (48.9%) was higher in the eukaryotic expression system than that observed in the bacterial system (27.6%). These results indicate that, in the investigated systems of heterologous cell-free protein expression, protein synthesis earns better solubility but lower production yield in the eukaryotic system, as compared to those in the bacterial system.

Bottom Line: On the one hand, elevated disorder content was associated with the increased ratio of soluble expression.On the other hand, overall propensity for detectable protein expression decreased with disorder content.Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Environmental Genomics, Kobe University, Nada 657-8501, Japan.

ABSTRACT
Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yields with the contents of intrinsic disorder bioinformatically predicted in the expressed sequences. This analysis revealed strong relationships between intrinsic disorder and protein amenability to heterologous cell-free expression. On the one hand, elevated disorder content was associated with the increased ratio of soluble expression. On the other hand, overall propensity for detectable protein expression decreased with disorder content. We further demonstrated that these tendencies are rooted in some distinct features of intrinsically disordered regions, such as low hydrophobicity, elevated surface accessibility and high abundance of sequence motifs for proteolytic degradation, including sites of ubiquitination and PEST sequences. Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

No MeSH data available.


Related in: MedlinePlus