期刊
PLANT BIOTECHNOLOGY
卷 39, 期 3, 页码 291-301出版社
JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY
DOI: 10.5511/plantbiotechnology.22.0628a
关键词
bFGF; Nicotiana benthamiana; protease-resistant; recombinant protein; transient production
资金
- Ministry of Education, Culture, Sports, Science and Technology (MEXT)
- Japan Science and Technology Agency, JST
- [VP29117939375]
This study demonstrates a high-level transient production system of functional PRbFGF in N. benthamiana leaves and an efficient tag-less purification technique of leaf crude extracts. The optimized Agrobacterium-mediated transient expression system achieved a 3-fold increase in bFGF production, and the introduction of a mutant protease-resistant version further doubled the yield. The purified PRbFGF showed comparable cell proliferation activity to E. coli-produced bFGF.
The human basic fibroblast growth factor (bFGF) is a protein that plays a pivotal role in cellular processes like cell proliferation and development. As a result, it has become an important component in cell culture systems, with applications in biomedical engineering, cosmetics, and research. Alternative production techniques, such as transient production in plants, are becoming a feasible option as the demand continues to grow. High-level bFGF production was achieved in this study employing an optimized Agrobacterium-mediated transient expression system, which yielded about a 3-fold increase in production over a conventional system. This yield was further doubled at about 185 mu g g-1 FW using a mutant protease-resistant version that degraded/aggregated at a three-fold slower rate in leaf crude extracts. To achieve a pure product, a two-step purification technique was applied. The capacity of the pure protease-resistant bFGF (PRbFGF) to stimulate cell proliferation was tested and was found to be comparable to that of E. coli-produced bFGF in HepG2 and CHO-K1 cells. Overall, this study demonstrates a high-level transient production system of functional PRbFGF in N. benthamiana leaves as well as an efficient tag-less purification technique of leaf crude extracts.
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