Protein Isoaspartate Methyltransferase Is a Multicopy Suppressor of Protein Aggregation in Escherichia coli
We used preS2-S′-β-galactosidase, a three-domain fusion protein that aggregates extensively at 43°C in the cytoplasm of Escherichia coli, to search for multicopy suppressors of protein aggregation and inclusion body formation and took advantage of the known differential solubility of preS2-S′-β-gala...
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| مؤلفون آخرون: | , , , , , |
| التنسيق: | article |
| منشور في: |
2005
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| الوصول للمادة أونلاين: | http://hdl.handle.net/10725/4167 http://dx.doi.org/10.1128/JB.187.4.1377-1383.2005 http://libraries.lau.edu.lb/research/laur/terms-of-use/articles.php http://jb.asm.org/content/187/4/1377.short |
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| _version_ | 1864513462936272896 |
|---|---|
| author | Kern, Renee |
| author2 | Malki, Abderrahim Abdallah, Jad Lebart, Jean-Claude Dubucs, Catherine Hee Yu, Myeong Richarme, Gilbert |
| author2_role | author author author author author author |
| author_facet | Kern, Renee Malki, Abderrahim Abdallah, Jad Lebart, Jean-Claude Dubucs, Catherine Hee Yu, Myeong Richarme, Gilbert |
| author_role | author |
| dc.creator.none.fl_str_mv | Kern, Renee Malki, Abderrahim Abdallah, Jad Lebart, Jean-Claude Dubucs, Catherine Hee Yu, Myeong Richarme, Gilbert |
| dc.date.none.fl_str_mv | 2005 2016-07-19T09:46:36Z 2016-07-19T09:46:36Z 2016-07-19 |
| dc.identifier.none.fl_str_mv | 0021-9193 http://hdl.handle.net/10725/4167 http://dx.doi.org/10.1128/JB.187.4.1377-1383.2005 Kern, R., Malki, A., Abdallah, J., Liebart, J. C., Dubucs, C., Yu, M. H., & Richarme, G. (2005). Protein isoaspartate methyltransferase is a multicopy suppressor of protein aggregation in Escherichia coli. Journal of bacteriology, 187(4), 1377-1383. http://libraries.lau.edu.lb/research/laur/terms-of-use/articles.php http://jb.asm.org/content/187/4/1377.short |
| dc.language.none.fl_str_mv | en |
| dc.relation.none.fl_str_mv | Journal of Bacteriology |
| dc.rights.*.fl_str_mv | info:eu-repo/semantics/openAccess |
| dc.title.none.fl_str_mv | Protein Isoaspartate Methyltransferase Is a Multicopy Suppressor of Protein Aggregation in Escherichia coli |
| dc.type.none.fl_str_mv | Article info:eu-repo/semantics/publishedVersion info:eu-repo/semantics/article |
| description | We used preS2-S′-β-galactosidase, a three-domain fusion protein that aggregates extensively at 43°C in the cytoplasm of Escherichia coli, to search for multicopy suppressors of protein aggregation and inclusion body formation and took advantage of the known differential solubility of preS2-S′-β-galactosidase at 37 and 43°C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43°C. First, we demonstrate that the differential solubility of preS2-S′-β-galactosidase results in a lactose-positive phenotype at 37°C as opposed to a lactose-negative phenotype at 43°C. We searched for multicopy suppressors of preS2-S′-β-galactosidase aggregation by selecting pink lactose-positive colonies on a background of white lactose-negative colonies at 43°C after transformation of bacteria with an E. coli gene bank. We discovered that protein isoaspartate methyltransferase (PIMT) is a multicopy suppressor of preS2-S′-β-galactosidase aggregation at 43°C. Overexpression of PIMT reduces the amount of preS2-S′-β-galactosidase found in inclusion bodies at 43°C and increases its amount in soluble fractions. It reduces the level of isoaspartate formation in preS2-S′-β-galactosidase and increases its thermal stability in E. coli crude extracts without increasing the thermostability of a control protein, citrate synthase, in the same extracts. We could not detect any induction of the heat shock response resulting from PIMT overexpression, as judged from amounts of DnaK and GroEL, which were similar in the PIMT-overproducing and control strains. These results suggest that PIMT might be overburdened in some physiological conditions and that its overproduction may be beneficial in conditions in which protein aggregation occurs, for example, during biotechnological protein overproduction or in protein aggregation diseases. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | LAURepo_b4e40764c904f0082fc92f252bb5de88 |
| identifier_str_mv | 0021-9193 Kern, R., Malki, A., Abdallah, J., Liebart, J. C., Dubucs, C., Yu, M. H., & Richarme, G. (2005). Protein isoaspartate methyltransferase is a multicopy suppressor of protein aggregation in Escherichia coli. Journal of bacteriology, 187(4), 1377-1383. |
| language_invalid_str_mv | en |
| network_acronym_str | LAURepo |
| network_name_str | Lebanese American University repository |
| oai_identifier_str | oai:laur.lau.edu.lb:10725/4167 |
| publishDate | 2005 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| spelling | Protein Isoaspartate Methyltransferase Is a Multicopy Suppressor of Protein Aggregation in Escherichia coliKern, ReneeMalki, AbderrahimAbdallah, JadLebart, Jean-ClaudeDubucs, CatherineHee Yu, MyeongRicharme, GilbertWe used preS2-S′-β-galactosidase, a three-domain fusion protein that aggregates extensively at 43°C in the cytoplasm of Escherichia coli, to search for multicopy suppressors of protein aggregation and inclusion body formation and took advantage of the known differential solubility of preS2-S′-β-galactosidase at 37 and 43°C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43°C. First, we demonstrate that the differential solubility of preS2-S′-β-galactosidase results in a lactose-positive phenotype at 37°C as opposed to a lactose-negative phenotype at 43°C. We searched for multicopy suppressors of preS2-S′-β-galactosidase aggregation by selecting pink lactose-positive colonies on a background of white lactose-negative colonies at 43°C after transformation of bacteria with an E. coli gene bank. We discovered that protein isoaspartate methyltransferase (PIMT) is a multicopy suppressor of preS2-S′-β-galactosidase aggregation at 43°C. Overexpression of PIMT reduces the amount of preS2-S′-β-galactosidase found in inclusion bodies at 43°C and increases its amount in soluble fractions. It reduces the level of isoaspartate formation in preS2-S′-β-galactosidase and increases its thermal stability in E. coli crude extracts without increasing the thermostability of a control protein, citrate synthase, in the same extracts. We could not detect any induction of the heat shock response resulting from PIMT overexpression, as judged from amounts of DnaK and GroEL, which were similar in the PIMT-overproducing and control strains. These results suggest that PIMT might be overburdened in some physiological conditions and that its overproduction may be beneficial in conditions in which protein aggregation occurs, for example, during biotechnological protein overproduction or in protein aggregation diseases.PublishedN/A2016-07-19T09:46:36Z2016-07-19T09:46:36Z20052016-07-19Articleinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article0021-9193http://hdl.handle.net/10725/4167http://dx.doi.org/10.1128/JB.187.4.1377-1383.2005Kern, R., Malki, A., Abdallah, J., Liebart, J. C., Dubucs, C., Yu, M. H., & Richarme, G. (2005). Protein isoaspartate methyltransferase is a multicopy suppressor of protein aggregation in Escherichia coli. Journal of bacteriology, 187(4), 1377-1383.http://libraries.lau.edu.lb/research/laur/terms-of-use/articles.phphttp://jb.asm.org/content/187/4/1377.shortenJournal of Bacteriologyinfo:eu-repo/semantics/openAccessoai:laur.lau.edu.lb:10725/41672021-03-19T10:00:45Z |
| spellingShingle | Protein Isoaspartate Methyltransferase Is a Multicopy Suppressor of Protein Aggregation in Escherichia coli Kern, Renee |
| status_str | publishedVersion |
| title | Protein Isoaspartate Methyltransferase Is a Multicopy Suppressor of Protein Aggregation in Escherichia coli |
| title_full | Protein Isoaspartate Methyltransferase Is a Multicopy Suppressor of Protein Aggregation in Escherichia coli |
| title_fullStr | Protein Isoaspartate Methyltransferase Is a Multicopy Suppressor of Protein Aggregation in Escherichia coli |
| title_full_unstemmed | Protein Isoaspartate Methyltransferase Is a Multicopy Suppressor of Protein Aggregation in Escherichia coli |
| title_short | Protein Isoaspartate Methyltransferase Is a Multicopy Suppressor of Protein Aggregation in Escherichia coli |
| title_sort | Protein Isoaspartate Methyltransferase Is a Multicopy Suppressor of Protein Aggregation in Escherichia coli |
| url | http://hdl.handle.net/10725/4167 http://dx.doi.org/10.1128/JB.187.4.1377-1383.2005 http://libraries.lau.edu.lb/research/laur/terms-of-use/articles.php http://jb.asm.org/content/187/4/1377.short |