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Expression and purification of recombinant Shiga toxin 2B from Escherichia coli O157:H7
- Source :
- African Journal of Biotechnology; Vol 15, No 21 (2016); 940-947
- Publication Year :
- 2016
- Publisher :
- Academic Journals, 2016.
-
Abstract
- Enterohemorrhagic Escherichia coli are important human food-borne pathogens. Recently, Shiga toxin-producing E. coli (STEC) causes life-threatening hemolytic-uremic syndrome (HUS). In this study, Stx2B gene, a subunit of Shiga toxin, was amplified via polymerase chain reaction (PCR) from the chromosomal DNA of clinical fecal sample using appropriate primers. The PCR product was cloned to commercially available plasmid pH6HTN His6HaloTag® T7 containing two purification tags, namely, six histadine tag and Halo tag. The integrity of the constructed plasmid was confirmed using restriction enzyme mapping and sequencing. Then, Stx2B protein expressed after induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) in E. coli JM109 (DE3) under the control of the T7 promotor. The two step purification trains were used to purify native Stx2B. First step purification was Ni-immobilized metal ion affinity chromatography (IMAC) column, followed by second step using HaloLink resin. The native Stx2B was obtained after column cleavage of halo-tag using HaloTEV protease. Maximum protein expression of Stx2B economically was obtained using 1 mM IPTG for 4 h at 37°C. Protein identity was confirmed by a band at ~11.4 kDa using 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and StxB2 yield was 450 µg ml-1 confirmed by Bradford assay. Recombinant Stx2B protein was produced in highly pure yield using HaloTag technology. Key words: Escherichia coli O157:H7, StxB gene, expression, HaloTag technology, purification.
- Subjects :
- 0301 basic medicine
medicine.disease_cause
Applied Microbiology and Biotechnology
law.invention
03 medical and health sciences
chemistry.chemical_compound
Plasmid
Affinity chromatography
law
Genetics
medicine
Escherichia coli O157:H7, StxB gene, expression, HaloTag technology, purification
Sodium dodecyl sulfate
Molecular Biology
Polyacrylamide gel electrophoresis
Bradford protein assay
Escherichia coli
biology
Shiga toxin
Molecular biology
030104 developmental biology
chemistry
biology.protein
Recombinant DNA
Agronomy and Crop Science
Biotechnology
Subjects
Details
- ISSN :
- 16845315
- Volume :
- 15
- Database :
- OpenAIRE
- Journal :
- African Journal of Biotechnology
- Accession number :
- edsair.doi.dedup.....c5d16961973ab47ff8a5facb635cf9ec