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Optimized expression conditions for enhancing production of two recombinant chitinolytic enzymes from different prokaryote domains

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Abstract

Enhancing functional gene expression is key to high-level production of active chitinases. For this purpose, the effects of culture cell density, inducer concentration, post-induction time and induction temperatures on the functional expression of two different chitinases (HsChiA1p, a family 18 archaeal chitinase and PtChi19p, a family 19 bacterial chitinase) were comparatively investigated. Results showed that the effect of each parameter on the activity of both chitinases was specific to each enzyme. In addition, different Escherichia coli host strains compatible with the expression in pET systems were assayed for active protein overexpression. When using BL21 Star (DE3), a significant increase of 60 % in expression was observed for the active archaeal chitinase HsChiA1p as compared to that found when using BL21 (DE3), indicating that the rne131 gene mutation efficiently stabilizes the mRNA for HsChiA1p. Using the Codon Adaptation Index value, rare codon analysis of the archaeal HschiA1 and bacterial Ptchi19 genes revealed that both DNA sequences were not optimal for maximal expression in E. coli. Different E. coli host strains possess extra copies of some of the tRNA genes for rare codons. For the Rosetta 2 (DE3) and the BL21 RP (DE3) strains, a significant increase of 40 % was reached for the activity of HsChiA1p and PtChi19p. Finally, as part of the protein still remained insoluble, the best conditions for recovering biologically active protein from inclusion bodies were established for each enzyme.

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Acknowledgments

G.-F., B. is grateful to Dr. Martha Clokie and Dr. Didier Philippe for allowing her to make a working visit to the Department of Infection, Immunity and Inflammation of the University of Leicester (United Kingdom) where she improved her knowledge and skills about protein expression. This work was supported by the Xunta de Galicia (Grant Number 10PXIB310278PR). G.-F., B. has a predoctoral fellowship from the Xunta de Galicia-Campus do Mar. L.-S., J. has a predoctoral fellowship from the University of Vigo.

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Authors and Affiliations

  1. Department of Functional Biology and Health Sciences, Microbiology Area, University of Vigo, Lagoas-Marcosende, 36310, Vigo, Spain

    Belén García-Fraga, Abigail F. da Silva, Jacobo López-Seijas & Carmen Sieiro

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  1. Belén García-Fraga
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  2. Abigail F. da Silva
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  3. Jacobo López-Seijas
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  4. Carmen Sieiro
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Correspondence to Carmen Sieiro.

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García-Fraga, B., da Silva, A.F., López-Seijas, J. et al. Optimized expression conditions for enhancing production of two recombinant chitinolytic enzymes from different prokaryote domains. Bioprocess Biosyst Eng 38, 2477–2486 (2015). https://doi.org/10.1007/s00449-015-1485-5

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  • DOI: https://doi.org/10.1007/s00449-015-1485-5

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