High-level transient production of a protease-resistant mutant form of human basic fibroblast growth factor in Nicotiana benthamiana leaves

Abstract

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 µg 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.

Keywords: Nicotiana benthamiana; bFGF; protease-resistant; recombinant protein; transient production.

Figure 1. T-DNA of Expression constructs and details of bFGF variants used. (A) Schematic representations of no signal peptide constructs that express 18 kDa bFGF in the form of the wild type (bFGF), GGG optimized (GGGbFGF) and protease-resistant (PRbFGF). The recombinant gene is under the control of a cauliflower mosaic virus 35S promoter (P35S) and Arabidopsis thaliana heat shock protein 18.2 terminator (HSPT). Other elements in the T-DNA are the translational enhancer Arabidopsis thaliana dehydrogenase untranslated region (AtADH 5′UTR) and the T-DNA left and right border (LB and RB) which marks the boundaries of the transgenes transferred during Agrobacterium-mediated transformation. (B) DNA sequence of GGG optimized bFGF. Underlined sequences are the identified GGGNDD or DGGGND whose GGG sequences were replaced by GGA. (C) Amino acid sequence alignment of wild type (WT) bFGF and protease-resistant bFGF (PR) showing replaced Cys⁷⁰ and Cys⁸⁸ to Ser⁷⁰ and Ser⁸⁸ respectively.

Figure 2. Optimization of agroinfiltration and extraction methods. (A) Time course of bFGF production in supplemented buffer vs. conventional buffer post infiltration. (B) Comparison of bFGF extracted when mixed for 10 min against sonication for 20 s on ice. (C) Optimization of NaCl concentration in the extraction buffer. (D) ELISA analysis of the optimal extraction buffer for bFGF in agroinfiltrated N. benthamiana leaf extracts. For panels A and D, the average values and the standard deviation from triplicate samples are shown. For panels B and C, positive control (PC) is 16.5 kDa Escherichia coli derived bFGF.

Figure 3. Comparison of transient bFGF production among variants in N. benthamiana leaf crude extracts. These include the wild type (bFGF), GGG optimized (GGGbFGF) and protease-resistant (PRbFGF) along with just empty vector (Empty) and E. coli derived bFGF (PC). (A) Western blot, (B) silver staining, and (C) ELISA. For panel C, data are shown as the mean±SD of triplicate measurements.

Figure 4. Stability analysis of PRbFGF and bFGF in crude extracts. (A) Western blot (upper panel) and CBB staining (lower panel) of bFGF in crude extracts, (B) western blot (upper panel) and CBB staining (lower panel) of PRbFGF in crude extracts, and (C) ELISA analysis. For panel C, average data was shown with the error bars representing the standard deviation from triplicate experiments.

Figure 5. Purification of PRbFGF using SP-Sepharose and heparin affinity chromatography. (A) Western blot, (B) silver staining. Included are the wild type crude extract (WT), the crude extract of PRbFGF agroinfiltrated leaves (CE), elution fraction after double purification (E) and the16.5 kDa E. coli derived bFGF (PC).

Figure 6. The effect of N. benthamiana produced PRbFGF on the cell proliferation of selected mammalian cell lines. (A) Hep G2 cells, and (B) CHO-K1 cells. For panels A and B, the mean from triplicate experiments were shown with the error bars representing the standard deviation.