[Cloning, expression of human keratinocyte growth factor and its purification and identification].
To clone KGF-2 gene, get hKGF-2 protein and detemine its activity. The cNDA of human KGF-2 was isolated from fetal lung by RT-PCR and cloned into pBV220 plasmid. The recombinant pBV220-hKGF-2 plasmid was transformed into E. coli (BL21), induced at 42 degrees C for the expression of hKGF-2. Recombinant human KGF-2 was purified from the ultrasonic-treated BL21 by heparin-Sepharose CL-6B treated column chromatography and cation exchange column chromatography. MTT method was used for the determination of its biological activity. SDS-PAGE showed that rhKGF-2 was expressed in E. coli BL21 as soluble protein of approximately 20kD. The rhKGF-2 protein can stimulate the proliferation of NIH3T3 cells significantly from 1 ng/mL to 10 ng/mL. HKGF-2 cDNA wasclned and highly expressed in E. coli BL21 and the purified rhKGF-2 showed the mitogenic activity on NIH3T3 cells.
Wu
Bin-Wen
BW
National Laboratory of Molecular Virology and Genetic Engineering, Institute of Virus Prevention and Control, Chinese Center of Disease Control, Beijing 100052, China.
Duan
Zhao-Jun
ZJ
Li
Wu-Ping
WP
Chen
Yong
Y
Lü
Hong-Liang
HL
Yi
Zuo-An
ZA
Zhang
Cheng-Hai
CH
Lin
Ju-Sheng
JS
Wang
Jia-Long
JL
Hou
Yun-De
YD
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005333:Fetus; D051526:Fibroblast Growth Factor 10 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008168:Lung / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D051526:Fibroblast Growth Factor 10; D011994:Recombinant Proteins