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Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin.
- Source :
-
Protein expression and purification [Protein Expr Purif] 2004 Oct; Vol. 37 (2), pp. 361-7. - Publication Year :
- 2004
-
Abstract
- An open reading frame of gelonin (Gel), one of ribosome inactivating proteins, was inserted into the vector pBSL-C which contains the coding region of chitin binding domain (CBD)-intein, resulting in the fusion expression of CBD-intein-Gel in Escherichia coli BL21 (DE3) by the induction of IPTG. The fusion product formed an aggregate of the misfolded protein, commonly referred to as inclusion bodies (IBs). The IBs were denatured and then refolded by step-wise dialysis. About 69% fusion protein was in vitro refolded to native state in the presence of GSSG and GSH as monitored by size-exclusion HPLC. The refolded CBD-intein-Gel was loaded onto chitin beads column equilibrated with 10 mM Tris buffer, 500 mM NaCl, pH 8.5, and about 2.4 mgGel/L culture with 96% homogeneity was directly eluted from the captured column by incubation at 25 degrees C under pH 6.5 for 48 h based on intein C-terminal self-cleavage. Western blot, ELISA, and in vitro inhibition of protein synthesis demonstrated that the bioactivity of recombinant Gel was comparable to that of native Gel purified from seeds. This implied that the purified Gel by this method is biologically active and suitable for further studies.
- Subjects :
- Animals
Blotting, Western
Chromatography, High Pressure Liquid
Cloning, Molecular
Electrophoresis, Polyacrylamide Gel
Enzyme-Linked Immunosorbent Assay
Escherichia coli enzymology
Escherichia coli metabolism
Hydrogen-Ion Concentration
Inhibitory Concentration 50
Isopropyl Thiogalactoside chemistry
Models, Chemical
Plasmids metabolism
Protein Conformation
Protein Denaturation
Protein Folding
Recombinant Fusion Proteins chemistry
Recombinant Proteins chemistry
Reticulocytes
Ribosome Inactivating Proteins, Type 1
Biochemistry methods
Inteins
Plant Proteins chemistry
Plant Proteins isolation & purification
Subjects
Details
- Language :
- English
- ISSN :
- 1046-5928
- Volume :
- 37
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Protein expression and purification
- Publication Type :
- Academic Journal
- Accession number :
- 15358358
- Full Text :
- https://doi.org/10.1016/j.pep.2004.06.037