[Establishment of purification procedure for recombinant fusion protein B7-2-PE40KDEL].
This study was aimed to establish downstream purification procedure by which the protein of interest can be purified to higher purity rapidly and efficiently. The different combinations of various purification strategies, methods and conditions were compared, including reversed phase chromatography, metal chelating chromatography, anion exchange chromatography, blue dye affinity chromatography, filtration chromatography and so on. The results showed that in reversed phase chromatography, isolated protein of interest was denatured and precipitated immediately after chromatography because methanol or acetonitrile were adopted as the organic phase. In blue dye affinity chromatography expecting to purify the protein of interest in one step, protein of interest was difficultly differentiated from mixed protein as much proteins bound to the chromatography media by non-specific affinity. While there is a translation-enhancing sequence T7-g10 in the PRSETA-B7-2-PE40KDEL expression vector, so it adds 6 histidines to the N terminus of the protein of interest, this allows to purify the protein of interest by metal chelating chromatography. Based on this characteristic, a three-step chromatography line including metal chelating chromatography, anion exchange chromatography and filtration chromatography was finally established after repeated experiments. By this way the purity of protein of interest reached 95% and the total recovery rate was 8%. The result of Western blot indicated that the expressed and purified recombinant B7-2-PE40KDEL could specifically bind with mAb against human B7-2 and multiple antibody against PEA. The cytotoxicity of the recombinant toxin tested by MTT method showed that the B7-2-PE40KDEL could selectively kill Jurkat cell line expressing CD28 receptor well and had no killing effect on the Raji cell line unexpressing CD28 receptor. It is concluded that a high efficient and speedy three-step purification procedure for the purifying recombinant protein B7-2-PE40KDEL was established, and this procedure possess selective killing activity on CD28 positive T lymphocytes.
Department of Immunology, Hospital Affiliated to Academy of Military Medical Sciences, Laboratory of Immunoassay, National Center of Biomedical Analysis, Beijing 100071, China.
Hai-Rong
HR
Guan
Yu-Ying
YY
Sun
Zhi-Hong
ZH
Yuan
Hui-Li
HL
Zhang
Fei
F
Liang
Nan
N
Liu
Si-Qi
SQ
Guo
Cai-Xia
CX
Xi
Yong-Zhi
YZ
Xi
D051940:B7-2 Antigen / Q000096:biosynthesis* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D018106:CD28 Antigens / Q000276:immunology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D051940:B7-2 Antigen; D001426:Bacterial Proteins; D018106:CD28 Antigens; C497964:CD86 protein, human; D011993:Recombinant Fusion Proteins; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa