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Identification, Characterization and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production.

Duwadi K, Chen L, Menassa R, Dhaubhadel S - PLoS ONE (2015)

Bottom Line: The yield of human therapeutic interleukin (IL)-10 produced in transgenic tobacco leaves was found to be below the critical level, and may be due to degradation by tobacco proteases.It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6.Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Western Ontario, London, ON, Canada.

ABSTRACT
Plants are an attractive host system for pharmaceutical protein production. Many therapeutic proteins have been produced and scaled up in plants at a low cost compared to the conventional microbial and animal-based systems. The main technical challenge during this process is to produce sufficient levels of recombinant proteins in plants. Low yield is generally caused by proteolytic degradation during expression and downstream processing of recombinant proteins. The yield of human therapeutic interleukin (IL)-10 produced in transgenic tobacco leaves was found to be below the critical level, and may be due to degradation by tobacco proteases. Here, we identified a total of 60 putative cysteine protease genes (CysP) in tobacco. Based on their predicted expression in leaf tissue, 10 candidate CysPs (CysP1-CysP10) were selected for further characterization. The effect of CysP gene silencing on IL-10 accumulation was examined in tobacco. It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6. Transient expression of CysP6 silencing construct also showed an increase in IL-10 accumulation in comparison to the control. Moreover, CysP6 localizes to the endoplasmic reticulum (ER), suggesting that ER may be the site of IL-10 degradation. Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

No MeSH data available.


Multiple sequence alignment of Peptidase_C1 (Papain) family candidate CysPs.Predicted amino acid sequences of a papain, NTCP23 (CysP1), CysP2, CysP4, CysP5, NTCP6 (CysP6), CysP7, CysP8 and CysP10 were aligned using CLUSTALW and shaded using BOXSHADE 3.21. Identical and conserved amino acids are shaded by dark and light grey, respectively. (*) indicates catalytic residues cysteine (C), histidine (H) and asparagine (N). (*) indicates the disulfide bridge forming cysteine residues. Red box indicates endoplasmic reticulum retention signal KDEL in CysP2 and CysP10. The conserved ERFNIN motif in the propeptide region is double-underlined. C-terminal amino acids of CysP6 and CysP8 beyond the positions 410 and 362, respectively, are not shown in the alignment.
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pone.0130556.g002: Multiple sequence alignment of Peptidase_C1 (Papain) family candidate CysPs.Predicted amino acid sequences of a papain, NTCP23 (CysP1), CysP2, CysP4, CysP5, NTCP6 (CysP6), CysP7, CysP8 and CysP10 were aligned using CLUSTALW and shaded using BOXSHADE 3.21. Identical and conserved amino acids are shaded by dark and light grey, respectively. (*) indicates catalytic residues cysteine (C), histidine (H) and asparagine (N). (*) indicates the disulfide bridge forming cysteine residues. Red box indicates endoplasmic reticulum retention signal KDEL in CysP2 and CysP10. The conserved ERFNIN motif in the propeptide region is double-underlined. C-terminal amino acids of CysP6 and CysP8 beyond the positions 410 and 362, respectively, are not shown in the alignment.

Mentions: To identify catalytic residues in the CysPs, the deduced amino acid sequences of papain family CysPs identified in this study were aligned, along with papain and a previously characterized tobacco CysP, NTCP23 (Fig 2). A catalytic triad comprised of Cys25, His159 and Asn175 (papain numbering) was found in the papain domain of all aligned CysPs. Enzymatic activity of a CysP is dependent on Cys25 and His159 residues, which exist as ion-pairs in a pH interval of 3.5–8 [42]. Besides the catalytic Cys residues, four other Cys residues were also identified to be conserved in the papain domain of all aligned CysPs. These residues are likely involved in disulfide bridge formation leading to proper folding and activity of the enzymes. The alignment shown in Fig 2 excludes CysP3 as it lacked several N-terminal amino acid residues, and critical residues Asn and His in the catalytic triad. A highly conserved block of amino acids interspersed with variable residues, EX3RX3FX2NX3I/VX3N (ERFNIN motif) is present in the propeptide region of all papain family CysPs [43].


Identification, Characterization and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production.

Duwadi K, Chen L, Menassa R, Dhaubhadel S - PLoS ONE (2015)

Multiple sequence alignment of Peptidase_C1 (Papain) family candidate CysPs.Predicted amino acid sequences of a papain, NTCP23 (CysP1), CysP2, CysP4, CysP5, NTCP6 (CysP6), CysP7, CysP8 and CysP10 were aligned using CLUSTALW and shaded using BOXSHADE 3.21. Identical and conserved amino acids are shaded by dark and light grey, respectively. (*) indicates catalytic residues cysteine (C), histidine (H) and asparagine (N). (*) indicates the disulfide bridge forming cysteine residues. Red box indicates endoplasmic reticulum retention signal KDEL in CysP2 and CysP10. The conserved ERFNIN motif in the propeptide region is double-underlined. C-terminal amino acids of CysP6 and CysP8 beyond the positions 410 and 362, respectively, are not shown in the alignment.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493103&req=5

pone.0130556.g002: Multiple sequence alignment of Peptidase_C1 (Papain) family candidate CysPs.Predicted amino acid sequences of a papain, NTCP23 (CysP1), CysP2, CysP4, CysP5, NTCP6 (CysP6), CysP7, CysP8 and CysP10 were aligned using CLUSTALW and shaded using BOXSHADE 3.21. Identical and conserved amino acids are shaded by dark and light grey, respectively. (*) indicates catalytic residues cysteine (C), histidine (H) and asparagine (N). (*) indicates the disulfide bridge forming cysteine residues. Red box indicates endoplasmic reticulum retention signal KDEL in CysP2 and CysP10. The conserved ERFNIN motif in the propeptide region is double-underlined. C-terminal amino acids of CysP6 and CysP8 beyond the positions 410 and 362, respectively, are not shown in the alignment.
Mentions: To identify catalytic residues in the CysPs, the deduced amino acid sequences of papain family CysPs identified in this study were aligned, along with papain and a previously characterized tobacco CysP, NTCP23 (Fig 2). A catalytic triad comprised of Cys25, His159 and Asn175 (papain numbering) was found in the papain domain of all aligned CysPs. Enzymatic activity of a CysP is dependent on Cys25 and His159 residues, which exist as ion-pairs in a pH interval of 3.5–8 [42]. Besides the catalytic Cys residues, four other Cys residues were also identified to be conserved in the papain domain of all aligned CysPs. These residues are likely involved in disulfide bridge formation leading to proper folding and activity of the enzymes. The alignment shown in Fig 2 excludes CysP3 as it lacked several N-terminal amino acid residues, and critical residues Asn and His in the catalytic triad. A highly conserved block of amino acids interspersed with variable residues, EX3RX3FX2NX3I/VX3N (ERFNIN motif) is present in the propeptide region of all papain family CysPs [43].

Bottom Line: The yield of human therapeutic interleukin (IL)-10 produced in transgenic tobacco leaves was found to be below the critical level, and may be due to degradation by tobacco proteases.It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6.Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Western Ontario, London, ON, Canada.

ABSTRACT
Plants are an attractive host system for pharmaceutical protein production. Many therapeutic proteins have been produced and scaled up in plants at a low cost compared to the conventional microbial and animal-based systems. The main technical challenge during this process is to produce sufficient levels of recombinant proteins in plants. Low yield is generally caused by proteolytic degradation during expression and downstream processing of recombinant proteins. The yield of human therapeutic interleukin (IL)-10 produced in transgenic tobacco leaves was found to be below the critical level, and may be due to degradation by tobacco proteases. Here, we identified a total of 60 putative cysteine protease genes (CysP) in tobacco. Based on their predicted expression in leaf tissue, 10 candidate CysPs (CysP1-CysP10) were selected for further characterization. The effect of CysP gene silencing on IL-10 accumulation was examined in tobacco. It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6. Transient expression of CysP6 silencing construct also showed an increase in IL-10 accumulation in comparison to the control. Moreover, CysP6 localizes to the endoplasmic reticulum (ER), suggesting that ER may be the site of IL-10 degradation. Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

No MeSH data available.