1. Optimization of heterologous expression of banana glucanase in E. coli
- Author
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Abughren Mohamed, Popović Milica, Dimitrijević Rajna, Burazer Lidija, Grozdanović Milica, Atanasković-Marković Marina, and Gavrović-Jankulović Marija
- Subjects
food allergen ,protein expression ,glucanase ,Chemistry ,QD1-999 - Abstract
For the heterologous production of a banana glucanase in Escherichia coli, its gene (GenBank GQ268963) was cloned into a pGEX-4T expression vector as a fusion protein with glutathione-S-transferase (GST). BL21 cells transformed with the GST-Mus a 5 construct were employed for the protein production induced by 1 mM of isopropyl-â-D-tiogalactopyranoside (IPTG). Conditions for the protein expression were optimized by varying the temperature (25°C, 30°C, and 37°C) and duration of protein expression (3h, 6h and 12h). The level of protein production was analyzed by densitometry of sodium dodecyl sulfate - polyacrylamide gel (SDS-PAG) after electrophoretic resolution of respective cell lysates. The optimal protein expression for downstream processing was obtained after 12h of cell growth under 25°C upon addition of IPTG. Recombinant GST-Mus a 5 purified by glutathione affinity chromatography revealed a molecular mass of about 60 kDa. The IgE and IgG reactivity of rGST-Mus a 5 was confirmed by dot blot analysis with individual patient’s sera from subjects with banana allergy and polyclonal rabbit antibodies against banana extract, respectively. Purified recombinant glucanase is a potential candidate for banana allergy diagnosis.
- Published
- 2012
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