The order and dis-order regions of the recombinant RT-L and RT-K were predicted by IUpred 3.0 (https://iupred3.elte.hu/). The statistical algorithm is used to predict the order and dis-order regions based on a 20 × 20 matrix, which characterizes the general preference of each pair of amino acids to be in contact as observed in a dataset of globular proteins (Erdős et al. 2021).

In-vitro studies Escherichia coli strains, growth kinetics, and transformation

Two strains of E. coli BL21(DE3) and BL21(shuffle) were used for protein expression. The recombinant plasmids were dissolved in sterile TE buffer (1 M Tris pH8, and 0.5 M EDTA) and then transformed into strains of the E. coli competent cells according to standard laboratory protocol (Hanahan et al. 1991). The transformed bacterial cells were cultured overnight in LB broth (supplemented with 100 µg/mL kanamycin) at 37 °C in a shaking incubator (180 g) for 16 h. The fresh LB (containing 100 µg/mL kanamycin) was inoculated and grown to reach an OD 600nm of 0.6, Isopropyl ß-D-1-thiogalactopyranoside (IPTG) was added at different concentrations (0.1, 0.5, and 1 mM). Additionally, samples were cultured at two different temperatures (20 °C and 37 °C), and ten post-induction times (2–20 h) to optimize the induction conditions.

SDS-PAGE analysis and western blotting

Cell pellets were dissolved in the lysis buffer (150 mM NaCl, 50 mM Tris-Cl pH8.0, 1.0% Triton X-100, and protease inhibitor cocktail (ab271306) and sonicated at 150 Hz amplitude with a 2 mm probe for 10 cycles each consisting of 20s of sonication followed by 10s intervals on ice. The sonication solution was centrifuged at 8500 g, at 4 °C for 15 min. The inclusion body and supernatant of cells were separately collected and the inclusion body part dissolved in 8 M urea. The protein of the inclusion body and supernatant were separately quantified by the Bradford assay. The size and the yield of recombinant RT-L and RT-K proteins were confirmed and compared by coomassie-blue stained 12% SDS-PAGE, respectively (Laemmli 1970). Western blot was done using mouse anti-His (Santa Cruz, USA) with a dilution ratio of 1:2500 and horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Sigma-Aldrich, Germany) with a dilution ratio of 1:4500. Finally, 3,3´-Diaminobenzidine (DAB, Sigma Aldrich, USA) was used to visualize the blots.

Purification and refolding of the recombinant RT-L and RT-K

The rRT-L and rRT-K were mostly produced in E. coli BL21 (shuffle) strain as inclusion body form. Also, rRT-K was found in the supernatant more than rRT-L. Both the insoluble and soluble fractions were allowed to pass through the Ni-NTA agarose column after equilibration (Qiagen-CN: 31014). The column was washed with five-bed volumes of the wash buffers (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M Urea, pH 6.3) three times. For the soluble fractions, the column was washed with another buffer containing (50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole, pH 8). Finally, the insoluble and soluble fractions were eluted with two-bed volumes of two different elution buffers (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M Urea pH 4.5 and 3) and (50 mM NaH2PO4, 300 mM NaCl, 200 and 250mM Imidazole pH 8), respectively. The concentration of purified recombinant proteins was quantified by the Bradford method (Bradford 1976). The salt was removed by dialysis buffer (5 mM DTT, 1 mM EDTA, 100 mM NaCl, 50 mM Tris-HCL pH 7.5, 0.1%V/V Triton X-100, 50% Glycerol, 5% W/V Sucrose, 4 mM L-Arginine, and protease inhibitor cocktail) for 24 h.

Analysis of rnase H and reverse transcriptase activity of RT-L and RT-K rnase H activity assay

The RNase H inactivity of rRT-K compared to rRT-L was properly assayed by the modified RT reaction in analytical techniques such as one-step RT-PCR and RT-LAMP. In this method, the SARS-CoV-2 RNA was converted to cDNA in the RT reactions as a template for rRT-L and rRT-K enzymes. The prepared cDNA was incubated for 30 min at 37 °C with DNase-I to degrade the first-strand DNA in the DNA-RNA hybrid. The treated cDNA was used in the one-step RT-PCR and RT-LAMP reactions and the product was visualized by electrophoresis and colorimetric way, respectively.

RT assay

The thermostable activity of rRT-L, rRT-K, and commercial RT (Add bio, Germany) was compared in the RT-PCR and RT-LAMP reactions. Therefore, SARS-CoV-2 RNA was used to prepare cDNA by the spike-specific reverse primer in the RT reaction. The variation parameters of the RT reaction were the temperature (50–60 °C) and incubation time (15, 20, and 30 min). The six products of the RT reaction of each rRT and commercial RT were used and compared in the PCR and LAMP reactions.

RT-PCR assay

The polymerase activities of rRT-L and rRT-K proteins were evaluated and compared with those using a commercial RT Assay Kit (Add Bio-Germany). Each 20-µL reaction contained 10 µL of 2x RT Reaction buffer, 2 µmol/L of reverse primers, 0.2–2 µL of each recombinant RT protein, and 7 µL of purified SARS-CoV-2 RNA, or 7 µL of nuclease-free H2O as non-template control (NTC). The reverse transcription was optimized to various parameters, including a range of temperatures (50–60 °C), incubation times (15, 20, and 30 min), and each rRT protein volume (0.2–4 ul). The PCR was performed with specific primers of the SARS-CoV-2 spike gene. The PCR program was as follows: 1 cycle at 95 °C for 5 min and 40 cycles including three steps at 95 °C for 30 s, 60 °C for 40 s, and 72 °C for 30 s, followed by a final extension step of 72 °C for 5 min. All reactions were done in a PCR system (ABI, USA), and results were analyzed from three technical repeats. The amplicons of respective fragments were analyzed via gel electrophoresis.

RT-LAMP assay

For the polymerase activity of recombinant RT proteins by the RT-LAMP analysis, the assays were carried out in triplicate and total reaction volumes of 10 µL. Briefly, 10 µL reactions were prepared to contain 1 µL of 10x Bst reaction buffer; 6 mM MgSO4; 1.4 mM each dATP, dGTP, dCTP, and dUTP; 1.6 µM each of primers of forward inner primer (FIP) and backward inner primer (BIP); 0.2 µM each of primers of F3 and B3; 0.4 µM each of forward loop (FL) and backward loop (BL) primers; 0.8 M betaine; 0.2 µL (1.6U) Bst3 (New England Biolabs, UK); 0.1 µL (0.1U) UNG (Biotechrabbit, Germany); 0.2 µL of each recombinant RT proteins, and 1 µL of purified SARS-CoV-2 RNA, or 1 µL of nuclease-free H2O as NTC. After adding 2 µL of SYBR green I diluted 1:100 in DMSO (Invitrogen-US) into the cap of each sample vial, the reaction mixture was set at 25 °C for 5 min and three different temperatures (55 °C, 60 °C, and 63.5 °C) for 60 min on a water bath, followed by an inactivation step of 85 °C for 5 min. Visualization of the product was detected by electrophoresis and by direct colorimetric ways too.

Statistical analyses

The SAS V.9.1 software was used for data analysis. To compare protein expression levels in the different conditions, full model two-way ANOVA was designed on the different factors including recombinant enzymes, strains, temperatures, and post-induction times. Also, the effect of interactions among treatments was computed. Multiple mean comparisons were done using the Tukey test and a p-value < 0.01 was usually chosen.