Transient expression, purification and characterization of bioactive human fibroblast growth factor 8b in tobacco plants |
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Authors: | Potula H H Surya Kumar Kathuria Sonal Roy Ghosh A K Maiti T K Dey S |
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Institution: | (1) Indian Institute of Technology, Kharagpur, India |
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Abstract: | 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.
Surya Kumar Potula and Sonal Roy Kathuria contributed equally to this work. |
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Keywords: | Bioactive FGF8b Molecular farming Vacuum infiltration Ni-NTA affinity chromatography MALDI-TOF-MS NIH3T3 cell proliferation·Glycosylation |
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