Hyper-expression of GFP-fused active hFGF21 in tobacco chloroplasts.

Human fibroblast growth factor 21 (hFGF21) is a promising candidate for metabolic diseases. In this study, a tobacco chloroplast transformation vector, pWYP21406, was constructed that consisted of codon-optimized encoding gene hFGF21 fused with GFP at its 5' terminal; it was driven by the promoter of plastid rRNA operon (Prrn) and terminated by the terminator of plastid rps16 gene (Trps16). Spectinomycin-resistant gene (aadA) was the marker and placed in the same cistron between hFGF21 and the terminator Trps16. Transplastomic plants were generated by the biolistic bombardment method and proven to be homoplastic by Southern blotting analysis. The expression of GFP was detected under ultraviolet light and a laser confocal microscope. The expression of GFP-hFGF21 was confirmed by immunoblotting and quantified by enzyme-linked immunosorbnent assay (ELISA). The accumulation of GFP-hFGF21 was confirmed to be 12.44 ± 0.45% of the total soluble protein (i.e., 1.9232 ± 0.0673 g kg-1 of fresh weight). GFP-hFGF21 promoted the proliferation of hepatoma cell line HepG2, inducing the expression of glucose transporter 1 in hepatoma HepG2 cells and improving glucose uptake. These results suggested that a chloroplast expression is a promising approach for the production of bioactive recombinant hFGF21.

Efficient expression of fusion human epidermal growth factor in tobacco chloroplasts.

BACKGROUND: Chloroplast transformation is a robust technology for the expression of recombinant proteins. Various types of pharmaceutical proteins including growth factors have been reported in chloroplasts via chloroplast transformation approach at high expression levels. However, high expression of epidermal growth factor (EGF) in chloroplasts with the technology is still unavailable. RESULTS: The present work explored the high-level expression of recombinant EGF, a protein widely applied in many clinical therapies, in tobacco chloroplasts. In this work, homoplastic transgenic plants expressing fusion protein GFP-EGF, which was composed of GFP and EGF via a linker, were generated. The expression of GFP-EGF was confirmed by the combination of green fluorescent observation and Western blotting. The achieved accumulation of the recombinant fusion GFP-EGF was 10.21 ± 0.27% of total soluble proteins (1.57 ± 0.05 g kg- 1 of fresh leaf). The chloroplast-derived GFP-EGF was capable of increasing the cell viability of the NSLC cell line A549 and enhancing the phosphorylation level of the EGF receptor in the A549 cells. CONCLUSION: The expression of recombinant EGF in tobacco chloroplasts via chloroplast transformation method was achieved at considerable accumulation level. The attempt gives a good example for the application of chloroplast transformation technology in recombinant pharmaceutical protein production.

Stable and efficient expression of human brain-derived neurotrophic factor in tobacco chloroplasts.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is an intensively studied neurotrophin that promotes various physiological processes, such as acceleration of cell proliferation and differentiation, and is, therefore widely used in clinical applications. METHODS AND RESULTS: In this study, an expression vector with a codon-optimized hBDNF gene was constructed and transferred into chloroplasts of tobacco by gene-gun. After three or four rounds of selection with optimal spectinomycin concentration, hBDNF was integrated into the chloroplast genome of homoplastomic plants, as confirmed by PCR and Southern hybridization. ELISA indicated that hBDNF fused with GFP represented approximately 15.72% ± 0.33% of total soluble protein in the leaves of transplastomic plants. Moreover, the chloroplast-derived hBDNF displayed biological activity similar to the commercial product. CONCLUSIONS: This is the first case report of hBDNF expression by chloroplast transformation in the plant model, providing an additional pathway for the production of chloroplast-expressed therapeutic proteins.

Successful production of human epidermal growth factor in tobacco chloroplasts in a biologically active conformation.

Human epidermal growth factor (hEGF) is an important therapeutic compound with multiple applications particularly in pharmaceutical industry. Human EGF has already been expressed in different expression systems, however, the production of hEGF with bioactivity in chloroplasts has not been successful so far. In this study, we expressed a 6 × His-tagged hEGF in tobacco chloroplasts in its native conformation for the potential of large-scale production of hEGF for industrial applications. Several transplastomic plant lines were obtained, which were screened by PCR (polymerase chain reaction) using primers specific to selectable gene aadA, hEGF- and GFP-coding sequences that were included in the chloroplast expression vector. The selected lines were confirmed to be homoplasmic by PCR verification and Southern blot analysis. Immunoblotting assays of homoplasmic lines using antibodies raised against hEGF confirmed the accumulation of hEGF in transplastomic plants and the ELISA results demonstrated the expression levels of hEGF were between 0.124% and 0.165% of the total soluble proteins (TSP), namely, 23.16-25.77 ng/g of the fresh weight. In terms of activity, the data from cell proliferation and elongation assays showed that the tobacco-derived recombinant hEGF was as bioactive as its commercial counterpart. To our knowledge, this is the first report of recombinant production of hEGF with native bioactivity form in the chloroplast stroma. Overall, our results demonstrate the potential of higher plant chloroplasts for the production of a human therapeutic, hEGF, in an active conformation.

Study on Protein Structures of Eight Mung Bean Varieties and Freeze-Thaw Stability of Protein-Stabilized Emulsions.

In order to evaluate the freeze-thaw stability of mung bean protein isolate (MPI)-stabilized emulsions and its relationship with protein structure, proteins of eight mung bean varieties were compared. The results revealed that MPIs prepared from all eight varieties were mainly composed of five subunit bands, with albumin and globulin content ranges of 188.4-310.3 and 301.1-492.7 mg/g total protein, respectively. Protein structural analysis revealed that random coil structure (32.34-33.51%) accounted for greater than 30% of MPI secondary structure. Meanwhile, analysis of protein properties revealed emulsifying activity index (EAI), emulsifying stability index (ESI) and flexibility value ranges of 6.735-8.598 m2/g, 20.13-34.25% and 0.125-0.182, respectively. Measurements of freeze-thaw stability of MPI emulsions demonstrated that exposures of emulsions to multiple freeze-thaw cycles resulted in significantly different emulsion creaming index, oiling-off, particle size and zeta potential values for the various emulsions. Moreover, the stabilities of all eight protein emulsions decreased with each freeze-thaw cycle, as demonstrated using optical micrographs. The correlation analysis method was used to study the correlation between the original structures, emulsifying properties of proteins and the freeze-thaw stability of MPI emulsions. Correlation analysis results revealed significant relationships between albumin content, subunit bands with a molecular weight of 26.9 kDa and emulsifying properties were significantly related to the freeze-thaw stability of MPI emulsion. Thus, by determining these indicator values, we can predict the freeze-thaw stability of MPI-stabilized emulsions.

Effects of treatment methods on the formation of resistant starch in purple sweet potato.

In order to determine the mechanisms underlying resistant starch formation, three treatments were used to prepare resistant starch from purple sweet potato. The resistant starch yield, amylose content, chain length distribution, thermal properties, and crystal structure were determined, and the results were compared with those of unmodified starch. Autoclaving, pullulanase, and pullulanase-autoclaving treatments significantly increased the resistant starch yield, amylose content, shorter amylopectin branch content, and gelatinisation temperatures of native purple sweet potato starch. Resistant starch prepared via pullulanase-autoclaving combination treatment exhibited the highest gelatinisation enthalpy value and the greatest degree of overall thermal stability. X-ray diffraction patterns and Fourier-transform infrared spectra analysis demonstrated that all three treatments transformed the starch crystalline structure from C-type to B-type, and no new groups were generated during the modification process; all the processes were only physical modifications.

Production of active human FGF21 using tobacco mosaic virus-based transient expression system.

Fibroblast growth factor (FGF) family has a wide range of metabolic processes. FGF21 exerts critical physiological functions in clinical application. This study aimed to explore a convenient and highly efficient approach for rhFGF21 expression using TMV-TES. Firstly, the vector pTTEV-GFP was constructed, followed by optimisation of the expression parameters in Nicotiana benthamiana. Then, the rhFGF21 encoding gene harbouring vector pTTEV-rhFGF21 was constructed. Agrobacterium-mediated vacuum infiltration was performed with the optimised parameters and the expression of rhFGF21 was confirmed by the immunoblotting analysis. ELISA revealed that the protein accumulation of rhFGF21 accounts for 0.11% of total soluble proteins. The biological activity was evaluated and the results suggested that tobacco-expressed rhFGF21 could stimulate the glucose uptake in swiss 3T3-L1 adipocytes, which was similar to the activity of commercial products, suggesting its native biological activity. Therefore, using TMV-TES to express rhFGF21 will be a feasible approach for the mass production of rhFGF21.