[Cloning, expression and identification of recombinant fusion protein GX1-rmhTNFalpha].
OBJECTIVE
To construct prokaryotic vector GX1-rmhTNF containing neovascular-targeting peptide GX1 and human TNFalpha and produce the GX1-rmhTNF protein.


METHODS
We have previously obtained a specific phage peptide CGNSNPKSC (GX1) binding to vasculature of human gastric cancer. The GX1-rmhTNFalpha vector was constructed by merging sequence of neovascular-targeting peptide GX1 with N-terminal of new recombined human TNFalpha (rmhTNFalpha) using gene engineering methods. The expression of the fusion protein GX1-rmhTNFalpha in E. coli was induced by temperature. The expression of GX1-rmhTNFalpha was detected by SDS-PAGE and Western blot.


RESULTS
A novel protein with expected molecular mass about 18,000 was found after SDS-PAGE and gel staining. The expressed product showed a good binding ability to anti-TNFalpha monoclonal antibody.


CONCLUSIONS
The prokaryotic vector GX1-rmhTNF was constructed and the expression of GX1-rmhTNF protein was successfully induced, which was helpful for further purification of GX1-rmhTNF protein.
Cao
Shan-shan
SS
State Key Laboratory of Cancer Biology, Institute of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. caoshansahn80@yahoo.com.cn
Wu
Kai-chun
KC
Yan
Zhen
Z
Wan
Yi
Y
Han
Yu
Y
Zhao
Li-na
LN
Fan
Dai-ming
DM
D015153:Blotting, Western; D003001:Cloning, Molecular; D016503:Drug Delivery Systems; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D013696:Temperature; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
D009842:Oligopeptides; D010455:Peptides; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C534327:cyclo(cysteinyl-glycyl-aspargyl-seryl-aspargyl-prolyl-lysyl-seryl-cysteine)