Expression of thermostable MMLV reverse transcriptase in Escherichia coli by directed mutation

Figures

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  Fig. 1. A The constructs including rRT-L and rRT-K genes based on the pET-9a expression vector. B Electrophoretic pattern of digested rRT-L and rRT-K constructs with XbaI and BamHI. C The alignment of two rRT-L and rRT-K sequences. Site point-directed mutations were highlighted

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  Fig. 2. Sequence optimization of native and mutant RT genes. A The optimized nucleic acid sequences were cloned in XbaI and BamHI sites of pET-9a. B The whole minimum free energy of the secondary structures, codon adaptation index (CAI), and GC content of non-optimized and optimized RT-K and RT-L messenger RNA. C The minimum free energy of 16 S rRNA docking with ribosome binding site (RBS) before and after RBS designing. D The probability of rRT-K and rRT-L solubility

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  Fig. 3. Molecular docking between rRT-L and rRT-K and Sars-CoV-2 RNA as receptor and ligand, respectively, using the HDOCK server

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  Fig. 4. The effect of site point-directed mutations on the structure stability (dis-order) of the recombinant RT-L. Prediction of structural stability before mutation (rRT-L) (A) and after mutation (rRT-K) (B)

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  Fig. 5. A, B Coomassie-blue stained SDS‐PAGE of rRT-L and rRT-K expression at 20 °C and 37 °C. Protein size markers (10–100 kDa) (Lane 1); supernatant fraction of cells lysate of E. coli BL21‐rRT-L (Lane 2); pellet fraction of cells lysate of E. coli BL21‐rRT-L (Lane 3); supernatant fraction of cells lysate of E. coli BL21-rRT-K (Lane 4); pellet fraction of cells lysate of E. coli BL21‐rRT-K (Lane 5); B negative control E. coli BL21(Lane 6). C, D pellet fraction of cells lysate of E. coli BL21 post-induction at 2, 4, 6, 8, 10, 12, 14, 16,18,20 h for both rRT-L and rRT-K. E Immunoblotting of unpurified recombinant proteins results using the anti‐His monoclonal antibody. Protein molecular weight marker (10–100 kDa) (Lane 1); before purification of rRT-L (Lane 2); before purification of rRT-K (Lane 3)

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  Fig. 6. Effect of temperature, and post-induction time on expression of the recombinant RT-L and RT-K enzymes in strain Shuffle (A) and DE3 (B). The error bars represent mean ± SD

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  Fig. 7. Purification of rRT-L and rRT-K. A Purified rRT-L and rRT-K samples with the native method were separated on 12% SDS-PAGE. Protein molecular weight marker (10–100 kDa) (Lane 1); unpurified lysate (Lane 2), eluted phase contains imidazole 200 mM (Lane 3), eluted phase contains imidazole 250mM (Lane 4). B Purified rRT-L and rRT-K samples with the denature method. Protein molecular weight marker (10–100 kDa) (Lane 1); unpurified lysate (Lane 2), eluted phase in pH3 (Lane 3), eluted phase in pH4.5 (Lane 4). C Immunoblotting of purified recombinant proteins results using the anti‐His monoclonal antibody. Protein molecular weight marker (10–100 kDa) (Lane 1); after purification of rRT-L (Lane 2); after purification of rRT-K (Lane 3)

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  Fig. 8. Eight site-directed point mutations in the connection and RNase H domains. A Five (438-442aa) and three (530, 568, and 659 aa) point mutations at the RT connection and RNase H active site domains, respectively. B In-silico analysis of RNase H active site sequence by the Phyre Investigator tool. The substitution of D530, E568, and D659 of rRT-L to R530, G568, and R659 amino acids in rRT-K, respectively, could inactive the RNase H domain but could not change its structure. Hot mutations amino acids (D530, E568, and D659) in the active site in the RNase H domain (Black arrow). The highest likelihood coming from mutations with long bars were candidates for substitutations (Orange arrow). Preferred residue (R530, G568, and R659) among the candidate mutants (Green arrow)

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  Fig. 9. RNase H activity assay. A The RNase H inactivity of rRT-K compared to rRT-L was assayed by the modified RT 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. B 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

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  Fig. 10. Effects of thermal treatment on the DNA polymerase activity of rRTs and their related RT-PCR as well as one-step RT-LAMP results. A Analysis of RT-PCR results of r-RT-L and rRT-K enzymes derived from E. coli at culture temperatures of 20 and 37 ºC. Lane 1,2: RT-PCR product of rRT-L at 20 and 37 °C culture temperature; Lane 3: Marker 100 bp; Lane 4: negative control (without RNA template); Lane 4,5: RT-PCR product of rRT-K at 20 and 37°C culture temperature. B–D The electrophoretic pattern of RT-PCR reactions was carried out with commercial RT, purified rRT-L, and rRT-K. Lane 1: Marker 100 bp; Lane 2: negative control (without RNA template); Lane 3,4,5: RT-PCR product at 50 °C, 55 °C, and 60 °C, respectively. E–G The one-step RT-LAMP product of the negative control sample (without RNA template), purified SARS-CoV-2 RNA derived from the patient’ swab was incubated with rRT-L, and rRT-K, respectively. The tubes and lanes numbered 1,2, and 3 point to one-step RT-LAMP product at 50 °C, 55 °C, and 60 °C, respectively. The positive results are marked with a blue arrow in the electrophoresis and colorimetric patterns