title: Optimized expression conditions for enhancing production of two recombinant chitinolytic enzymes from different prokaryote domains.
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.
authors.forename: Belén
authors.initials: B
authors.lastname: García-Fraga
authors.forename: Abigail F
authors.initials: AF
authors.lastname: da Silva
authors.forename: Jacobo
authors.initials: J
authors.lastname: López-Seijas
authors.forename: Carmen
authors.initials: C
authors.lastname: Sieiro
mesh_terms: D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D019617:Halobacterium salinarum / Q000201:enzymology*; D031161:Pseudoalteromonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases