Open AccessOptimization of heterologous expression of banana glucanase in E. coli
Author(s) -
Mohamed Abughren,
Milica Popović,
Rajna Dimitrijević,
Lidija Burazer,
Milica Grozdanović,
Marina Atanasković–Marković,
Marija Gavrović-Jankulović
Publication year - 2012
Publication title -
journal of the serbian chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.227
H-Index - 45
eISSN - 1820-7421
pISSN - 0352-5139
DOI - 10.2298/jsc110309158a
Subject(s) - microbiology and biotechnology , polyclonal antibodies , recombinant dna , lac operon , escherichia coli , expression vector , western blot , fusion protein , heterologous expression , chemistry , glucanase , biochemistry , gel electrophoresis , molecular mass , biology , polyacrylamide gel electrophoresis , antibody , gene , enzyme , immunology
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