Learning about protein solubility from bacterial inclusion bodies

Martínez-Alonso, Maira; González-Montalbán, Núria; García‐Fruitós, Elena; Villaverde, Antonio

doi:10.1186/1475-2859-8-4

Cited by 70 publications

(46 citation statements)

References 59 publications

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“…On the other hand, the overall mGFP specific fluorescence in the presence of DnaK/DnaJ was lower than in the absence of these chaperones. This is consistent with recent observations reporting that higher protein yield in a production process necessarily results in a decrease of conformational quality (10,16,18). To determine whether the positive effect of DnaK and DnaJ in assisting protein folding could be extended to the expression of other recombinant proteins in insect cells, we also tested coexpression of these proteins with foot-and-mouth disease virus (FMDV) VP1 and VP2 capsid proteins and human alphagalactosidase A.…”

supporting

confidence: 58%

Rehosting of Bacterial Chaperones for High-Quality Protein Production

Martínez-Alonso

Toledo-Rubio

Noad

et al. 2009

Appl Environ Microbiol

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Coproduction of DnaK/DnaJ in Escherichia coli enhances solubility but promotes proteolytic degradation of their substrates, minimizing the yield of unstable polypeptides. Higher eukaryotes have orthologs of DnaK/ DnaJ but lack the linked bacterial proteolytic system. By coexpression of DnaK and DnaJ in insect cells with inherently misfolding-prone recombinant proteins, we demonstrate simultaneous improvement of soluble protein yield and quality and proteolytic stability. Thus, undesired side effects of bacterial folding modulators can be avoided by appropriate rehosting in heterologous cell expression systems.

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supporting

confidence: 58%

Rehosting of Bacterial Chaperones for High-Quality Protein Production

Martínez-Alonso

Toledo-Rubio

Noad

et al. 2009

Appl Environ Microbiol

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“…4 bacteria, the most common protein production platform, often yields recombinant polypeptides totally or partially deposited as inclusion bodies. 5 although recent studies have shown that inclusion bodies might contain functional protein species suitable for in situ enzymatic reactions, [6][7][8][9][10][11][12] protein production processes are in general aimed at soluble species. therefore, inclusion bodies are separated by differential sedimentation and further discarded.…”

mentioning

confidence: 99%

Protein Aggregation and Soluble Aggregate Formation Screened by a Fast Microdialysis Assay

et al. 2010

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Protein aggregation is a major obstacle in recombinant protein production as it reduces the yield of soluble polypeptides. Also, the formation of aggregates occurring in the soluble fraction is more common than formerly expected, and the prevalence of these entities might significantly affect the average quality of the soluble protein species. Usually, the formation of soluble aggregates remains unperceived because analytical methods such as dynamic light scattering are not routinely applied as quality control procedures. The authors have developed a methodologically simple and fast procedure, based on microdialysis and image processing, that reveals the aggregation tendency of a given protein in a specific environment. Because they also show a good correlation between macroscopic aggregation and soluble aggregate formation, the microdialysis approach also permits an estimation of the occurrence of soluble aggregates.

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“…BL21-DE3-pLysS was the selected strain for large scale expression because of its capacity to suppress the basal expression of the T7 promoter due to the production of T7 lysozyme, a natural inhibitor of T7 RNA polymerase. The extraction of the expressed protein from E. coli BL21-DE3-pLysS cells demonstrated that the MP was present only in the insoluble fraction, probably as the result of inclusion bodies formation (Martínez-Alonso et al, 2009). Similar cases of insolubility of proteins expressed in E. coli system were reported with the replicases of Tobacco mosaic virus (TMV) and Citrus tristeza virus (CTV) (Hills et al, 1987;Çevik et al, 2008) and with TMV MP (Chen et al, 2000).…”

mentioning

confidence: 53%

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Calegario¹,

Labate²,

Peroni³

et al. 2012

Trop. plant pathol.

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Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plantvector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

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Learning about protein solubility from bacterial inclusion bodies

Cited by 70 publications

References 59 publications

Rehosting of Bacterial Chaperones for High-Quality Protein Production

Rehosting of Bacterial Chaperones for High-Quality Protein Production

Protein Aggregation and Soluble Aggregate Formation Screened by a Fast Microdialysis Assay

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

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