High-level expression and purification of Tat-haFGF19-154.
Human acidic fibroblast growth factor (haFGF) stimulates repair and regeneration of central and peripheral nerves after various injuries. However, it is unable to cross the blood-brain barrier (BBB). To produce a therapeutic haFGF with cell-permeable activity, we fused the haFGF(19-154) gene with Tat-PTD. After its construction by a single-step insertion of a polymerase chain reaction (PCR)-amplified coding sequence, the vector pTat-haFGF(19-154)-His was expressed in Escherichia coli BL21 (DE3) cells. The optimal expression level of the soluble fusion protein was up to 36.7% of the total cellular protein. The recombinant Tat-haFGF(19-154)-His was purified by a combination of Ni-NTA affinity, Sephadex G-25, and heparin affinity chromatography to 95% as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final yield was 171 mg/l culture. Purified Tat-haFGF(19-154)-His had distinct mitogenic activity in Balb/c 3T3 cells, as measured by methylthiazoletetrazolium (MTT) assay and its ED(50) was 3.931 x 10(-4) micromol/l. Tat-haFGF(19-154)-His protein intravenously injected at the dose of 10 mg/kg could be detected in the pallium and hippocampi.
Huang
Yadong
Y
Biopharmaceutical Research & Development Center, Jinan University College of Pharmacy, Guangzhou, Guangdong, 510632, People's Republic of China.
Rao
Yulan
Y
Feng
Chengli
C
Li
Yanmei
Y
Wu
Xiaoping
X
Su
Zhijian
Z
Xiao
Jian
J
Xiao
Yechen
Y
Feng
Wenke
W
Li
Xiaokun
X
D000818:Animals; D041702:BALB 3T3 Cells; D049109:Cell Proliferation; D002540:Cerebral Cortex / Q000737:chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000493:pharmacokinetics; D015870:Gene Expression; D015696:Gene Products, tat / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000493:pharmacokinetics; D006624:Hippocampus / Q000737:chemistry; D006801:Humans; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000493:pharmacokinetics; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; C000595303:Tat-haFGF (19-154); D054322:tat Gene Products, Human Immunodeficiency Virus; D005346:Fibroblast Growth Factors