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.
Anti-HBV assay
The anti-HBV assay was performed as previously described [23] 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 [24], and the solubility of these proteins was determined using Predict Protein [25]. 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.
Structure modeling
The structure of the protein was predicted by fold recognition methodology using the I-TASSER [29,30,31] and Phyre2 [32] 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 [35] 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 [36].
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.