Construction and application of a prokaryotic vector which expresses the protein that can be quickly purified by IMAC.
A vector was constructed by inserting a pair of complementary oligo nucleotides encoding 6 histidine residues into the polylinker's upstream of the prokaryotic high expression vector pBV220. The resultant vector is named pBV222. Proteins expressed by this vector will have a 6-histidine tail as an affinity handle fused to their N-terminus and can be quickly purified by one-step immobilized metal affinity chromatography (IMAC). This plasmid was verified by restriction mapping and DNA sequencing. When GM-CSF and IL-2 cDNA were closed into pBV222, expressed proteins in the inclusion body showed the predicted molecular weight and biological activity. The expressed bacteria were dissolved in 6 mol/L guanidine.HCl and the supernatant was loaded directly to IMAC. IL-2 and GM-CSF fusion proteins were eluted by the pH gradient, and over 90% purity was achieved.
Li
F
F
National Laboratory of Molecular Virology and Genetic Engineering, CAPM, Beijing, China.
Gong
H
H
Li
Y
Y
Zhang
Z
Z
Hou
Y
Y
D002846:Chromatography, Affinity / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013997:Time Factors
D018076:DNA, Complementary; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor