[CDNA cloning of human leptin and its expression].
To clone cDNA of human leptin gene and obtain leptin protein for future study on leptin binding proteins. The cDNA of human leptin with 6 x his-tag was cloned by over-hang extension PCR protocol using human genomic DNA as template, and subcloned into in vitro expression vector pIVEX2.3MCS, and the fusion protein was expressed in vitro by Rapid Translation System (RTS) (RTS500 cycle primer Kit and RTS500 ProteoMaster of Roche company). The apparent molecular weight(19.46 kD) and the immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot, and the expressed fusion protein stayed mainly in the supernatant of the reaction mixture in soluble form. This work provides us solid basis for further study on new leptin-associated proteins.
Jia
Zhen-Yu
ZY
Zhejiang Academy of Medical Sciences, Hangzhou 310013, China. zhenyujia@yahoo.com
Fu
Xiao-Min
XM
Jin
Ai-Hua
AH
Cao
Jiang
J
D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D020738:Leptin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D020738:Leptin; D011993:Recombinant Fusion Proteins