Transient expression, purification and characterization of bioactive human fibroblast growth factor 8b in tobacco plants.
cDNA of human fibroblast growth factor 8 isoform b (FGF8b) was cloned for the first time into a plant expression vector with or without endoplasmic reticulum retention signal (KDEL) and was transiently expressed as His tagged fusion protein in Nicotiana tabacum leaves through Agrobacterium mediated gene transfer by vacuum infiltration method. Expression of FGF8b was confirmed by ELISA and Western blot using anti-FGF8b antibody and the expression level was measured as 4.1% of total soluble protein of tobacco leaves. The expressed recombinant protein was purified by Ni-NTA affinity chromatography and its molecular weight was determined by MALDI-TOF-MS. Schiff's test, Concanavalin A (Con A) immunoblot and enzymatic deglycosylation indicated that the high molecular mass was due to glycosylation of the FGF8b expressed in plant cells. Measurement of its biological activity in NIH3T3 cells by thymidine incorporation and MTT assay showed induction of cell proliferation. These results indicate that biologically active recombinant FGF8b could be expressed in tobacco plants.
Potula
H H Surya Kumar
HH
Indian Institute of Technology, Kharagpur, India.
Kathuria
Sonal Roy
SR
Ghosh
A K
AK
Maiti
T K
TK
Dey
S
S
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D051524:Fibroblast Growth Factor 8 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D041681:NIH 3T3 Cells; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism*; D014170:Transformation, Genetic
D017931:DNA Primers; C496456:FGF8 protein, human; D011994:Recombinant Proteins; D051524:Fibroblast Growth Factor 8