Expression of novel fusion antiviral proteins ricin a chain-pokeweed antiviral proteins (RTA-PAPs) in Escherichia coli and their inhibition of protein synthesis and of hepatitis B virus in vitro
Ricin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a protein synthesis inhibition activity assay.
Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with a 50% protein synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized protein ricin A chain mutant-Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on protein synthesis inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance.
Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on protein synthesis inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies.
KeywordsFusion proteins Ricin Pokeweed antiviral protein Hepatitis B virus Antiviral agent Ribosome-inactivating proteins
Half-maximal response concentration of a drug
100% protein synthesis inhibitory concentration
50% protein synthesis inhibitory concentration
Pokeweed antiviral protein isoform 1 from leaves
Pokeweed antiviral proteins
Pokeweed antiviral protein isoform 1 from seeds
Ricin A Chain
Pokeweed antiviral proteins (PAPs) are expressed in several organs of the plant pokeweed (Phytolacca Americana) and are potent type I Ribosome Inactivating Proteins (RIPs). Their sizes vary from 29-kDa to 30-kDa and are able to inhibit translation by catalytically removing specific adenine residues from the large rRNA of the 60S subunit of eukaryotic ribosomes [1, 2, 3]. Furthermore, PAPs can depurinate specific guanine residues, in addition to adenine, from the rRNA of prokaryotic ribosomes. PAPs possess antiviral activity on a wide range of plant and human viruses through various mechanisms . Transgenic plants expressing different forms of PAPs were found to be resistant to various viral and fungal infections [4, 5]. The anti-viral activity of PAPs against human viruses has been described against Japanese encephalitis virus , human immunodeficiency virus-1 (HIV-1) , human T-cell leukemia virus-1 (HTLV-1) , herpes simplex virus (HSV) , influenza , hepatitis B virus (HBV) , and poliovirus . PAPs low to moderate cytotoxicity to non-infected cells, in contrast to infected cells, makes PAPs very attractive candidates in the development of potential therapeutics and as protective agents against pathogens in transgenic plants.
Ricin is expressed in the seeds of the castor oil plant (Ricinus communis) and is one of the most potent type II RIPs. It is highly toxic to mammalian cells as its A chain can efficiently be delivered into the cytosol of cells through the mechanism of its B chain. The B chain serves as a galactose/N-acetylgalactosamine binding domain (lectin) and is linked to the A chain via disulfide bonds . Ricin can induce 50% apoptosis in mammalian cells at concentrations below 1 ng/mL while showing no to low activity on plant and E. coli ribosomes. It is important to note however that the ricin A chain (RTA) on its own has less than 0.01% of the toxicity of the native protein in a cell culture test system. It was furthermore shown that RTA alone had no activity on non-infected and tobacco mosaic virus (TMV)-infected tobacco protoplasts alike. RTA lacks the ability to enter the cell without the action of the B chain . RTA depurinates a universally conserved adenine residue within the sarcin/ricin loop (SRL) of the 28S rRNA to inhibit protein synthesis. Though there are currently no commercially available therapeutic applications, RTA is extensively studied in the development of immunotoxins .
The therapeutic potential of PAPs and RTA has been explored for over thirty years, though dosage dependant side effects have limited clinical applications. These proteins have shown very low cytotoxicity to non-infected cells; however, PAPs administration in mouse models has resulted in hepatic, renal and gastrointestinal tract damage with a median lethal dose (LD50) as low as 1.6 mg/Kg . Interestingly, RTA shows no toxicity even at high doses with similar half-life times. Nevertheless, all RIPs show immunosuppressive effects to various degrees. Many studies have described the various dose-limiting side effects of these proteins when used as immunotoxins (i.e. vascular leak syndrome, hemolytic uremic syndrome and pluritis, among others) [17, 18]. Nonetheless, some patients achieved complete or partial remission against Refractory B-Lineage Acute Lymphoblastic Leukemia with sub-toxic dosages, for example.
Fusion and hybrid proteins of RTA and PAPs have also been developed in pursuit of selectively targeting infected cells and selectively recognizing viral components, though with limited success [19, 20]. Indeed, the engineering of novel therapeutic fusion proteins with higher specificity, selectivity, and potency with fewer side effects is a leading strategy in drug development that is more often than not limited by our still new understanding of protein structure and function. Another limiting factor is the availability of efficient protein structure prediction and simulation software. It is only with the recent advent of more sophisticated in silico tools that protein engineering became a viable alternative to conventional drug discovery techniques.
Based on the data gathered on these two proteins over the last thirty years and the newly available in silico tools, the authors hypothesized that it is possible to create novel fusion proteins between RTA and PAPs that will be more effective than either of the proteins alone at sub-toxic dosages against specific infectious diseases and that will be cheaper to produce than available therapeutics. The purpose of this research is thus to assess the potential production amenability and sub-toxic antiviral activities of newly created and biologically active novel fusion proteins between RTA and PAPs [21, 22].
Here, we describe the development of an effective and scalable production system in Escherichia coli and of purification methods that enabled accurate determination of RTA-PAPs protein synthesis inhibition in vitro. We similarly describe the in vitro reduced cytotoxicity and significant anti-HBV activity of RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) by detecting HBV DNA in the supernatant of HepAD38 cells. The reengineering of RTA-PAPS1 into RTA mutant- Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) to improve its production in Escherichia coli and to enhance its gain of function is also described using the most up-to-date protein structure and function prediction software available online.
E. coli in vivo expression system and rabbit reticulate lysate protein synthesis inhibition
Design of the DNA sequences of the proteins for E. coli in vivo expression system
The two cDNA sequences coding for RTA-PAPS1 (541 amino acids) and for RTAM-PAP1 (556 amino acids including the N terminal 6-His tag) were optimized for E. coli expression and chemically synthesized by AscentGene.
E. coli in vivo expression vector
The cDNA coding for RTA-PAPS1 and RTAM-PAP1 sequences described above were generated by PCR using the primers RP1-A48 (5’TTTAACTTTAAGAAGGAGATATACATATGATCTTCCCGAAACAGTACC) or RPAP1-A48 (5’TTTAACTTTAAGAAGGAGATATACATATGCACCACCATCACCACCATA) and RPAP1-B50 (5’CAGCCGGATCTCAGTGGTGGTGCTCGAGTTAGGTAGTCTGGCAAGAACCG). Each PCR fragment was then subcloned into the E. coli pET30a expression vector (Novagene) between the NdeI and XhoI restriction endonuclease sites to generate the pET30a-RP1 and pET30a-6H-RPAP1 vectors respectively. The inserts were validated by DNA sequencing.
E. coli in vivo protein production
The above described vectors were transformed into E. coli BL21(DE3) cells (NEB) and expression of the proteins were examined from individual clones and analyzed by either Western blot using a monoclonal antibody specific to ricin A chain (ThermoFisher, RA999) or SDS gel stained with Comassie blue (ThermoFisher). Optimal conditions were determined and protein production induced in the presence of 1 mM IPTG from 1 L culture for each protein. The bacteria were then harvested by centrifugation, followed by lysing the cell pellets with 50 ml of lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 0.2% Triton X100 and 0.5 mM EDTA). After sonication (3x2min), the soluble lysates were recovered by centrifugation at 35 K rpm for 40 min. The insoluble pellets were further extracted with 40 ml of 6 M Urea and the inclusion bodies (IB) were recovered by centrifugation at 16 K rpm for 20 min. Clarified IB were then dissolved with 20 ml of buffer 8b (proprietary formulation of AscentGene). The soluble proteins were then recovered by centrifugation (please contact the authors for more details).
E. coli protein purification
Ricin-PAPS1 proteins were purified by gel filtration column (Superdex 200 from GE Healthcare) under denaturing condition (6 M Urea). Peak fractions were pooled and powder Guanidine was added to a concentration of 5 M for complete denaturing. Denatured Ricin-PAPS1 was then added dropwise to the refolding buffer (50 mM Tris-Cl, pH 8.1, 0.4 M L-Arginine, 0.5 mM oxidized glutathione and 5 mM reduced glutathione) for refolding. The solution was stirred at room temperature for 10 min before allowing the refolding reaction to be further carried out at 4 °C for > 20 h. Clarified and refolded Ricin-PAPS1 proteins were then concentrated before going through the endotoxin removal process and the ammonium sulfate precipitation step. The resulting mixture was dialyzed in the formulation buffer containing 20 mM HEPES-Na, pH 7.9, 20% glycerol, 100 mM NaCl, 2.5 mM tris(2-carboxyethyl)phosphine (TCEP) and 1 mM EDTA.
The purification of the native RTAM-PAP1 from soluble lysate was achieved by affinity versus His-tag on Ni-sepharose column (GE Healthcare). After extensive washes with the lysis buffer, loosely bound proteins were eluted with the lysis buffer containing 40 mM Imidazole (I40). RTAM-PAP1 proteins were eluted with the elution buffer (20 mM Tris-Cl, pH 7.9, 100 mM NaCl, 1 mM EDTA and 300 mM Imidazole). A second purification step using Hydroxylapatite column (GE Healthcare) was used to further separate RTAM-PAP1 from co-purified host proteins. A third purification step, gel filtration on a fast protein liquid chromatography (FPLC) column of Superose 12 (GE Healthcare), was necessary to completely get rid of degraded and/or premature protein products. The resulting mixture was dialyzed in the formulation buffer containing 20 mM HEPES-Na, pH 7.9, 200 mM NaCl, 0.2 mM CaCl2 and 0.5 mM EDTA.
Rabbit reticulate lysate protein synthesis inhibition
The inhibitory activities of RTA-PAPS1 and RTAM-PAP1 were tested by using the Rabbit Reticulate Lysate TnT® Quick Coupled Transcription/Translation System and the Luciferase Assay System (Promega). Briefly, each transcription/translation reaction was performed according to the instructions for use (IFU) in the presence of a T7 Luciferase reporter DNA, and the Luciferase expression level was determined with a Wallac Microplate Reader. Transcription/translation runs were done twice with and without addition of five different concentrations of RTA-PAPS1 and RTAM-PAP1 in order to determine the inhibitory effect of the proteins. RTA-PAPS1 and RTAM-PAP1 concentrations were adjusted by taking sample purity into consideration.
The anti-HBV assay was performed as previously described  with the modification of using HepAD38 cells by ImQuest BioSciences. ImQuest BioSciences developed a multi-marker screening assay utilizing the HepAD38 cells to detect proteins, RNA, and DNA intermediates characteristic of HBV replication. The HepAD38 cells are derived from HepG2 stably transfected with a single cDNA copy of hepatitis B virus pregenomic RNA, in which HBV replication is regulated by tetracycline. Briefly, HepAD38 cells were plated in 96-well flat bottom plates at 1.5 × 104 cells/well in Dulbecco’s modified Eagle’s medium supplemented with 2% FBS, 380 μg/mL G418, 2.0 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.1 mM nonessential amino acids (ThermoFisher). After 24 h, six ten fold serial dilutions of RTA-PAPS1 prepared in the same medium were added in triplicate. Lamivudine (3TC from Sigma Aldrich) was used as the positive control, while media alone was added to cells as a negative control (virus control, VC). Three days later, the culture medium was replaced with fresh medium containing the appropriately diluted RTA-PAPS1. Six days following the initial administration of RTA-PAPS1, the cell culture supernatant was collected, diluted in qPCR dilution buffer, and then used in a real-time quantitative qPCR assay using a Bio-Rad CFX384 Touch Real-Time PCR Detection System. The HBV DNA copy number in each sample was interpolated from the standard curve by the supporting software. A tetrazolium dye uptake assay (ThermoFisher) was then employed to measure cell viability, which was used to calculate cytotoxic concentration (TC50).
Protein design optimization
Physiochemical profiling and specific structural features
The molecular profile of the protein was determined using the Protparam tool of ExPASy , and the solubility of these proteins was determined using Predict Protein . The presence of disulfide bonds was determined using the DiANNA 1.1 webserver [26, 27, 28]. Functional effects of point mutations were determined using SNAP2 of Predict Protein.
The structure of the protein was predicted by fold recognition methodology using the I-TASSER [29, 30, 31] and Phyre2  prediction servers. The determined protein structures were then validated by Verify 3D [33, 34]. The quality of the structure was determined using the QMEAN6 program of the SWISS-MODEL  workspace.
Design of RTAM-PAP1
Three major changes were made to RTA-PAPS1 in order to increase its solubility, its efficacy against infected cells and to further reduce its toxicity.
Firstly, two point mutations, as predicted by SNAP2 of Predict Protein to have the least effect on function, were introduced into the RTA moiety to replace the Cysteine (Cys) residues with Alanine residues in order to completely avoid unwanted disulfide bond formation at position 171 and 259 (C171A and C259A) to create RTA mutant (RTAM).
Secondly, the natural semi-flexible linker previously used was replaced with a newly designed soluble flexible G rich linker with a rigid CASP2 recognition site (GGGGSDVADI(GGGGS)2) to allow better autonomous function of each moiety with minimal steric hindrance and to further enhance the chimeric protein’s ability to induce cell apoptosis .
Thirdly, A different variant than PAPS1 was used, PAP1, retrieved from National Centre for Biotechnology Information database (NCBI) with access number P10297.2 in order to further enhance activity against HBV and further reduce toxicity of the chimeric protein.
Lastly, a 6-His tag was added at the N terminal of the protein RTAM-PAP1 in order to minimize effect on structure and function and to increase native protein recovery from E. coli production.
Production and purification of recombinant RTA-PAPS1 in E. coli culture
Inhibitory activity of recombinant RTA-PAPS1 in the rabbit reticulate lysate TnT® system
Anti-HBV evaluation of recombinant RTA-PAPS1 in HepAD38 cells
Protein design optimization
Production and purification of recombinant RTAM-PAP1 in E. coli culture
Inhibitory activity of recombinant RTAM-PAP1 vs. RTA-PAPS1 in the rabbit reticulate lysate TnT® system
The chimeric protein RTA-PAPS1 was expressed only in inclusion bodies with very little solubility, except under heavy denaturing conditions. The refolding process was successful to a certain extent as more than one conformation was observed. This was probably due to the two free Cysteine residues in RTA and to the nature of the semi-flexible linker, which allowed the close proximity of Cys at position 260 to the Cys residues at position 364 and 385 (confirmed by DiANNA 1.1 webserver and I-Tasser). The addition of TCEP was necessary and a difference in bioactivity (> 2 fold) was observed between samples (results not shown). RTA-PAPS1 with the addition of TCEP was very bioactive and with a noticeable synergetic activity between RTA and PAPS1, which was probably limited by steric hindrance once again due to the nature of the semi-flexible quality of the linker. This was confirmed during the anti-HBV assays. The significant anti-HBV activity of RTA-PAPS1 was apparent and probably due to the ability of both moieties to depurinate rRNA but also polynucleotide, single-stranded DNA, double stranded DNA and mRNA [11, 39]. HBV is a double stranded DNA reverse transcriptase virus. The observed higher standard deviations for RTA-PAPS1 compared to those observed for 3TC between samples at the same concentration during the anti-HBV assay were probably due to the presence of different conformations of the same protein. Additionally, dubious results were seen at the highest concentration of 600 nM for cytotoxicity, which were probably due to an unwanted reaction that occurs when the protein buffer solution to growth media ratio is high (> 30% at 600 nM). Furthermore, it was observed that the steric hindrance diminished the ability of RTA-PAPS1 to penetrate infected cells by > 2 fold compared to PAPS1 alone (the activity of RTA-PAPS1 on other viruses such as HIV-1 for example was found to be much lower than that of PAPS1 alone, results not shown). It was for these reasons that the decision to redesign the fusion protein with PAP1 and a new linker was taken. The decision to engineer an entirely new linker was taken despite the slight possibility that the natural semi-flexible linker used might have been the reason for the significant anti-HBV activity observed. Indeed, the natural semi-flexible linker might have allowed the fusion protein to adopt a unique conformation with a novel or improved existing anti-HBV activity.
The fusion protein RTAM-PAP1 expression went very well as we almost exclusively obtained native protein production with high solubility (barely any in inclusions bodies). It was however necessary to use a three step purification protocol in order to obtain soluble proteins with > 90% homogeneity. Nonetheless, 0.1 mg of protein at > 95% purity and 0.22 mg of protein at > 90% purity were obtained from 1 L of culture. This yield is probably explained by the increased toxicity of PAP1 to E. coli compared to that of PAPS1 (> 10 fold) . The bioactivity of RTAM-PAP1 was increased, much more than expected with very little to no sign of steric hindrance. The introduction of the two point mutations in the RTA moiety and of the flexible linker really made a difference in solubility and activity. Also, perhaps, fine-tuning the formulation buffer to better preserve protein integrity allowed for optimum activity. The synergetic effect of both moieties was very apparent and probably due to the fact that RTA and PAP1 do not dock onto the ribosome at the same site and, thus, led to a reduction of partially depurinated and still functional ribosomes .
The chimeric proteins combining RTA and PAPs are potent novel anti-viral proteins with gain of function in protein synthesis inhibition activity and anti-HBV activity in vitro with minimal cytotoxicity. The introduction of two point mutations in RTA and of a flexible linker greatly improved solubility and activity. RTAM-PAP1 can be overexpressed, recovered and purified from soluble lysate. It is expected that the anti-viral properties of RTAM-PAP1 against plant and animal pathogens will be even greater than that of either RTA-PAPS1 or PAPs with even lesser general toxicity. Indeed, this should be true as long as the original natural semi-flexible linker used did not have an unforeseen positive effect on function of RTA-PAPS1. For these reasons, it is the opinion of the authors that a full characterization of RTAM-PAP1 activity against chronic HBV infection be done both in vitro and, if successful, in vivo as it is a potential potent new therapeutic that can be produced at low costs to be used as a standalone or in combination with existent therapies.
This work was funded by Ophiuchus Medicine which owns the rights to the patents pending of the fusion proteins described in this study. The entire study was done at the request of, by and for Ophiuchus Medicine. The funding body participated in the design, optimization and data generation of the fusion proteins and linkers in silico and for analyzing the generated data. The funding body participated in conception, design and data analysis regarding the protein production and purification system and protein inhibition assays and antiviral assays. The funding body participated in writing the manuscript.
Availability of data and materials
All data generated or analyzed during this study are included in this published article, further details or raw data are available from the corresponding author on reasonable request.
YH was responsible for the design, optimization and data generation of the fusion proteins and linkers in silico and for analyzing the generated data. YH made substantial contributions to conception, design and data analysis regarding the proteins production and purification system and protein inhibition assays and antiviral assays. YH was a major contributor in writing the manuscript. SH supervised the design of the fusion proteins and was involved in the conception, design and data analysis regarding the proteins production and purification system and protein inhibition assays and antiviral assays. SH revised the entire manuscript’s content critically. HG was responsible for the conception, design and data generation regarding the proteins production and purification system and protein inhibition assays. HG was involved in drafting and revising critically some of the content of the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors are either directly or indirectly affiliated to Ophiuchus Medicine. Y.H. and S.O. are officers and shareholders of Ophiuchus Medicine. H.G.’s AscentGene is a subcontractor of Ophiuchus Medicine. This work was funded by Ophiuchus Medicine which owns the rights to the patents pending of the fusion proteins described in this study. The entire study was done at the request of, by and for Ophiuchus Medicine. The funding body participated in the design, optimization and data generation of the fusion proteins and linkers in silico and for analyzing the generated data. The funding body participated in conception, design and data analysis regarding the protein production and purification system and protein inhibition assays and antiviral assays. The funding body participated in writing the manuscript.
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