Recombinant pebulin protein, a type 2 ribosome-inactivating protein isolated from dwarf elder (Sambucus ebulus L.) shows anticancer and antifungal activities in vitro.

In this study, encoding sequence of a new type 2 RIP (pebulin) was isolated and cloned from dwarf elder (Sambucus ebulus L.) native to the northern regions of Iran. The nucleotide sequence of pebulin was ligated to the pET-28a(+) expression plasmid and cloned into the E. coli strain BL21 (DE3) in order to express heterologously of recombinant protein. The recombinant pebulin protein was mainly produced in the form of insoluble inclusion bodies probably because to absence of N-glycosylation process in E. coli. Therefore, in order to increase the expression of recombinant protein in soluble form, co-expression of the target protein with the pG-Tf2 chaperone plasmid and incubation of bacterial culture under low temperature were used to enhance solubility and accumulation of recombinant protein. After purification of the recombinant protein using affinity chromatography method, the bioactivity of pebulin was analyzed by hemagglutination, anticancer, and antifungal assays. The results of the hemagglutination assay showed that purified pebulin agglutinated erythrocytes in all human blood groups. In addition, pebulin considerably inhibited the proliferation of cancer cell lines MCF-7 and HT-29 in a time- and dose-dependent manner and indicated remarkably growth-inhibiting effect against the plant pathogenic fungi such as Alternaria solani and Fusarium oxysporum.

Fusion to elastin-like polypeptide increases production of bioactive human IFN-γ in tobacco.

The plant-based expression systems are now accredited as bioreactors for the high production of various biopharmaceuticals. However, low levels of agglomeration and the absence of effective procedures for purification of recombinant proteins have remained two essential obstacles in molecular farming. In this research, we have studied the production of human interferon gamma (hIFN-γ) in tobacco and analyzed the effects of elastin-like polypeptide (ELP) tag and subcellular localization on its accumulation. We report a remarkable enhancement of accumulation of the fusion proteins versus the corresponding unfused hIFN-γ proteins. Furthermore, the hIFN-γ (with and without ELP) accumulated to higher levels in the endoplasmic reticulum. The ELP fusion proteins were successfully recovered from total soluble protein with adding 2.75 M NaCl and three rounds of inverse transition cycling (ITC). The hIFN-γ was also separated from ELP with Enterokinase cleavage of the fusion protein and recovered by ITC. Inverse transition analysis indicated that the hIFN-γ-ELP variants aggregate above their inverse transition temperature and at high ionic strength. Investigation of glycosylation revealed that fused or unfused hIFN-γ proteins are N-glycosylated in different cellular locations. Moreover, N-glycosylation analysis and bioassay showed that fusion to ELP does not disturb glycosylation process and antiviral activity of hIFN-γ.

Production of bioactive human IFN-γ protein by agroinfiltration in tobacco.

In animals, interferon-γ (IFN-γ) is known as a cytokine involved in antiviral and anticancer activities with a higher biochemical activity in contrast to other IFNs. To produce recombinant human IFN-γ (hIFN-γ) protein in tobacco, factors influencing gene delivery were first evaluated for higher efficiency of transient expression by fluorometric measurement of GUS activity. Higher levels of transient expression were observed in leaves of Nicotiana tabacum cv. Samsun infiltrated with GV3101 strain (optical density equal to 1.0 at 600 nm) under treatment of 200 µM AS at 4 days post agroinfiltration (dpa). The Samsun cv. proved to be amenable with 1.4- and 1.5-fold higher levels of transient expression than Xanthi and N. benthamiana, respectively. In addition, the GV3101 remained the best strain for use in transient assays without any necrotic response in tobacco. The levels of transient hIFN-γ expression were also estimated in the Samsun cv. infiltrated with different Agrobacterium tumefaciens strains carrying various expression constructs. Higher levels of accumulation were obtained with targeting the hIFN-γ protein to endoplasmic reticulum (ER) or apoplastic space than those expressed into cytoplasm. Moreover, antiviral bioassay revealed that recombinant hIFN-γ protein produced in tobacco is biologically active and protects the Vero cells from infection generated by vesicular stomatitis virus (VSV).

Elastin-like polypeptide fusions for high-level expression and purification of human IFN-γ in Escherichia coli.

In this study, the ELP sequence was fused to human interferon-γ (hIFN-γ) and hIFN-γ-ELP fusion protein accumulated with high levels of yield and purity, compared with the corresponding unfused hIFN-γ protein. The hIFN-γ was exclusively produced in the form of insoluble inclusion bodies while the hIFN-γ was relatively soluble when expressed as an ELP fusion protein. The insoluble inclusion bodies were then solubilized under denaturing conditions, refolded in the presence of arginine and purified by single-step ion-exchange chromatography. The fusion to ELP signidficantly increased the accumulation of hIFN-γ by 10-fold with a stable expression on average of 46.85% of total soluble protein (TSP). Furthermore, three rounds of Inverse Transition Cycling (ITC) purification increased overall purity of the hIFN-γ-ELP to 98 ±â€¯5%. The recovery amount of the fusion protein found to be dependent on the NaCl concentration, with increase of NaCl concentration, a greater fraction of the hIFN-γ-ELP was aggregated. However, due to the presence of an aliphatic guest residue in ELP sequence, the high concentration of salt was necessary to trigger the inverse phase transition of hIFN-γ-ELP fusion protein. Moreover, recombinant hIFN-γ and hIFN-γ-ELP proteins purified from E. coli possessed a relatively similar bioactivity based on viral cytopathic assay.

Isolation and functional characterization of two thioredoxin h isoforms from grape.

Thioredoxins (Trxs) are small ubiquitous proteins that participate in dithiol-disulfide exchange reactions. In contrast to animals and prokaryotes, plants possess different types of Trxs that play a vital role in a number of different cellular processes. Two full-length cDNAs encoding different Trx h isoforms, designated VvTrx h2 and VvTrx h3, were isolated and cloned from grape (Vitis vinifera L. cv. Askari) berry tissue by rapid amplification of cDNA ends (RACE) method. VvTrx h2 and VvTrx h3 were heterologously expressed in Escherichia coli and their activities were compared using DTT-dependent insulin reduction and 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) reduction activities. The NADPH-dependent DTNB reduction assay demonstrated that the both VvTrx h isoforms were reduced by NADPH-dependent thioredoxin reductase (NTR) from E. coli. Under heat shock treatment, the recombinant VvTrx h proteins formed the oligomeric structures at above 50 °C with a decrease in their disulfide reductase activities. The redox-dependent structural changes of VvTrx h2 and VvTrx h3 revealed that their oligomeric structures were changed into monomers and significantly increased their disulfide reductase activities. Furthermore, the both recombinant proteins were able to conserve a DTNB reduction activity even after 15 min heating at 99 °C.