Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin.
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.
Guo
Chenyun
C
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, PR China.
Li
Zhuoyu
Z
Shi
Yawei
Y
Xu
Mingqun
M
Wise
John G
JG
Trommer
Wolfgong E
WE
Yuan
Jingming
J
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D047668:Inteins; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D008956:Models, Chemical; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012156:Reticulocytes; D054789:Ribosome Inactivating Proteins, Type 1
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D007544:Isopropyl Thiogalactoside; C025674:GEL protein, Gelonium multiflorum