Expression, purification, and characterization of recombinant protein GX1-rmhTNFalpha.
A phage-displayed peptide CGNSNPKSC (GX1) was obtained previously in our lab, which could specifically bind to the vasculature of human gastric cancer. GX1-rmhTNFalpha was a fusion protein of GX1 and recombinant mutant human tumor necrosis factor alpha (rmhTNFalpha), which was designed by us with the expectation of enhancing selectivity of rmhTNFalpha. The DNA fragment encoding GX1 was cloned into the vector pBV220 with rmhTNFalpha between the EcoRI site and the BamHI site, and then expressed in Escherichia coli DH5alpha by temperature induction. Subsequently, E. coli DH5alpha was lysed, and the GX1-rmhTNFalpha protein was found in both soluble form and inclusion bodies. The protein was fractionated with ammonium sulfate deposition from 30% to 60%, and purified by cation and anion exchange chromatography using SP Sepharose Fast Flow column and Q Sepharose Fast Flow column. The purity of protein was then identified by SDS-PAGE and HPLC. Subsequent studies showed that GX1-rmhTNFalpha had high bioactivity of 5.65 x 10(8) IU/ml, which was similar with natural human TNFalpha and could reach the tumor site relatively faster than rmhTNFalpha.
State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, Shaanxi Province, People's Republic of China.
Shanshan
S
Cao
Yan
Y
Liu
Xiaohua
X
Li
Yingqi
Y
Zhang
Jun
J
Wang
Wenqi
W
Du
Yu
Y
Han
Haifeng
H
Jin
Lina
L
Zhao
Kaichun
K
Wu
Daiming
D
Fan
D000818:Animals; D004926:Escherichia coli / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009928:Organ Specificity; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000493:pharmacokinetics*; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000493:pharmacokinetics*; D013274:Stomach Neoplasms / Q000378:metabolism*; D014018:Tissue Distribution; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000493:pharmacokinetics*
D010455:Peptides; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha