Comparing the Yield of Recombinant Human Factor VII Protein Expressed by the rDNA-Promoter with the CMV-Promoter in Iranian Lizard Leishmania
Abstract
Background: Iranian Lizard Leishmania (I.L.L) is a nonpathogenic Leishmania strain. Due to its advantages, several recombinant proteins have been produced in this host. However, I.L.L shows a lower yield of recombinant protein expression compared to other commercial hosts. Considering the role of protease enzymes in protein digestion, we selected cysteine protease B (CPB) to investigate its impact on recombinant protein yield in I.L.L.
Methods: we generated gene knockouts by utilizing homologous recombination (HR) and CRISPR methods. To assess the efficacy of the designed construct, we compared the yield of recombinant human factor VII (rhFVII) production between cells transfected with the pLEXSY-hyg2-FVII vector and the CMV-promoter-based construct (pF7cmvneo).
Results: The knockout of a single CPB gene allele through the HR method or the complete knockout of all alleles through the CRISPR method led to cell death. This outcome suggests that even the deletion of a single CPB gene allele diminishes the protein to a level insufficient for the survival of I.L.L, indicating a critical dependency on the presence of this protein for the organism's viability. rhFVII exhibited a greater expression yield with the pLEXSY construct compared to the pF7cmvneo construct in I.L.L. The lower expression rate of pF7cmvneo may be influenced by epigenetic factors related to the CPC gene or the RNA polymerase used for the expression of that promoter.
Conclusion: Therefore, considering alternative integration targets for CMV-promoter-based constructs and incorporating UTR sequences of I.L.L high-expression proteins in the vector may enhance recombinant protein expression rates.
1. Abdi Ghavidel A, Jajarmi V, Bandehpour M, Kazemi B. Polycistronic Expression of Multi-Subunit Complexes in the Eukaryotic Environment: A Narrative Review. Iran J Parasitol. 2022;17(3):286-95.
2. Klatt S, Simpson L, Maslov DA, Konthur Z. Leishmania tarentolae: Taxonomic classification and its application as a promising biotechnological expression host. PLoS Negl Trop Dis. 2019;13(7):e0007424.
3. de Oliveira TA, Silva Wd, da Rocha Torres N, et al. Application of the LEXSY Leishmania tarentolae system as a recombinant protein expression platform: A review. Process Biochemistry. 2019;87:164-73.
4. Breitling R. The LEXSY platform for recombinant protein expression. Farm animal proteomics Wageningen Academic Publishers. 2013.
5. Dortay H, Mueller-Roeber B. A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker. Microb Cell Fact. 2010;9:29.
6. Kazemi, B, Moazzen F, Abadi A, Ghadjari A. Isolation a lizard Leishmania promastigote from its natural hostin Iran. Journal of Biological Sciences. 2004;4(5):620-623
7. Abdi Ghavidel A, Aghamiri S, Jajarmi V, Bandehpour M, Kazemi B. The Influence of Different Culture Media on the Growth and Recombinant Protein Production of Iranian Lizard Leishmania Promastigote. Iran J Parasitol. 2022;17(4):543-53.
8. Ehya F, Kalantari S, Bandehpour M, Kazemi B. Preliminary Information of Iranian Lizard Leishmania Promastigote Transcriptome Sequencing by Next Generation Sequencing (NGS) Method. Iran J Parasitol. 2023;18(3):362-368.
9. Yadegari Z, Bandehpour M, Kazemi B, Sharifi-Sarasiabi K. Expression of Recombinant Human Amelogenin in Iranian Lizard Leishmania and Its Biological Function Assay. Iran J Public Health. 2015;44(7):987-96.
10. Taromchi AH, Kazemi B, Mahmazi S, Bandehpour M. Heterologous Expression of Human IL-29 (IFN-Î) in Iranian Lizard Leishmania. Iran J Biotechnol. 2013;11(3):168-74.
11. Donyavi T, Bandehpour M, Kazemi B. Preparation of transgenic Iranian lizard Leishmania coding HIL-12. Iran J Microbiol. 2017;9(5):305-11.
12. Huang LF, Sinaga DS, Tan CC, Hsieh SJM, Huang CH. Expression of Recombinant Human Octamer-Binding Transcription Factor 4 in Rice Suspension Cells. Int J Mol Sci. 2021;22(3):1409.
13. Kim NS, Kim TG, Kim OH, et al. Improvement of recombinant hGM-CSF production by suppression of cysteine proteinase gene expression using RNA interference in a transgenic rice culture. Plant Mol Biol. 2008;68:263-75.
14. Duwadi K, Chen L, Menassa R, Dhaubhadel S. Identification, Characteriz-ation and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production. PLoS One. 2015;10(7):e0130556.
15. Kazemirad E, Reisi Nafchi H, Latifi A, Raoofian R, Mohebali M, Hajjaran H. Comparison of Cysteine Protease B Gene Expression between Clinical Isolates of Leishmania tropica, Leishmania major and Leishmania infantum. J Med Microbiol Infect Dis. 2019;7(3):72-8.
16. Buxbaum LU, Denise H, Coombs GH, Alexander J, Mottram JC, Scott P. Cysteine Protease B of Leishmania mexicana Inhibits Host Th1 Responses and Protective Immunity 1. J Immunol. 2003;171(7):3711-7.
17. Scala A, Micale N, Piperno A, et al. Targeting of the Leishmania mexicana cysteine protease CPB2.8ΔCTE by decorated fused benzo thiophene scaffold. RSC Adv. 2016;6(36):30628-35.
18. Suhr ST, Ramachandran R, Fuller CL, Veldman MB, Byrd CA, Goldman D. Highly-restricted, cell-specific expression of the simian CMV-IE promoter in transgenic zebrafish with age and after heat shock. Gene Expr Patterns. 2009;9(1):54-64.
19. Mirzaahmadi S, Asaadi-Tehrani G, Bandehpour M, et al. Expression of recombinant human coagulation factor VII by the Lizard Leishmania expression system. J Biomed Biotechnol. 2011; 2011:873874.
20. Zhang WW, Lypaczewski P, Matlashewski G. Application of CRISPR/Cas9-Mediated Genome Editing in Leishmania. In: Michels PAM, Ginger ML, Zilberstein D, editors. Trypanosomatids: Methods and Protocols. New York, NY: Springer US; 2020. p.199-224.
21. Zhang WW, Matlashewski G, Singh U. Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in Leishmania. mSphere. 2019;4(4):e00408-19.
22. Denise H, McNeil K, Brooks DR, Alexander J, Coombs GH, Mottram JC. Expression of multiple CPB genes encoding cysteine proteases is required for Leishmania mexicana virulence in vivo. Infect Immun. 2003;71(6):3190-5.
23. Hide M, Bras-Gonçalves R, Bañuls AL. Specific cpb copies within the Leishmania donovani complex: evolutionary interpretations and potential clinical implications in humans. Parasitology. 2007; 134(Pt 3):379-89.
24. Cruz A, Beverley SM. Gene replacement in parasitic protozoa. Nature. 1990; 348(6297): 171-3.
25. Dean S, Sunter J, Wheeler RJ, Hodkinson I, Gluenz E, Gull K. A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open Biol. 2015;5(1):140197.
26. Zhang WW, Matlashewski G. CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani. mBio. 2015;6(4):e00861.
27. Negreira GH, Monsieurs P, Imamura H, et al. High throughput single-cell genome sequencing gives insights into the generation and evolution of mosaic aneuploidy in Leishmania donovani. Nucleic Acids Res. 2021;50(1):293-305.
28. Duncan SM, Jones NG, Mottram JC. Recent advances in Leishmania reverse genetics: Manipulating a manipulative parasite. Mol Biochem Parasitol. 2017;216:30-8.
29. Níttolo AG, Bañuelos CP, Saborit JI, Tekiel V, Sánchez DO, Levy GV. TbRRM1 knockdown produces abnormal cell morphology and apoptotic-like death in the bloodstream form of T. brucei. Mol Biochem Parasitol. 2018;224:1-5.
30. Huynh TT, Huynh VT, Harmon MA, Phillips MA. Gene Knockdown of γ-Glutamylcysteine Synthetase by RNAi in the Parasitic Protozoa Trypanosoma brucei Demonstrates That It Is an Essential Enzyme. J Biol Chem. 2003;278(41):39794-800.
31. Abdi Ghavidel A, Aghamiri S, Raee P, et al. Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Leishmania Strains. Acta Parasitol. 2024; 69(1):121-134.
32. Alves CR, Côrte-Real S, DeFreitas Rosa M, GiovanniDe-Simone S. Detection of cysteine-proteinases in Leishmania amazonensis promastigotes using a cross-reactive antiserum. FEMS Microbiol Lett. 2000;186(2):263-7.
33. Casgrain PA, Martel C, McMaster WR, Mottram JC, Olivier M, Descoteaux A. Cysteine Peptidase B Regulates Leishmania mexicana Virulence through the Modulation of GP63 Expression. PLoS Pathog. 2016;12(5):e1005658.
34. Raymond F, Boisvert S, Roy G, et al. Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species. Nucleic Acids Res. 2012;40(3):1131-47.
35. Kushnir S, Gase K, Breitling R, Alexandrov K. Development of an inducible protein expression system based on the protozoan host Leishmania tarentolae. Protein Expr Purif. 2005;42(1):37-46.
36. Maree JP, Patterton HG. The epigenome of Trypanosoma brucei: a regulatory interface to an unconventional transcriptional machine. Biochim Biophys Acta. 2014;1839(9):743-50.
37. Castro Neto AL, Brito A, Rezende AM, Magalhães FB, de Melo Neto OP. In silico characterization of multiple genes encoding the GP63 virulence protein from Leishmania braziliensis: identification of sources of variation and putative roles in immune evasion. BMC Genomics. 2019;20(1):118.
38. Bifeld E, Lorenzen S, Bartsch K, Vasquez JJ, Siegel TN, Clos J. Ribosome Profiling Reveals HSP90 Inhibitor Effects on Stage-Specific Protein Synthesis in Leishmania donovani. mSystems. 2018; 3(6):e00214-18.
39. Azevedo A, Toledo JS, Defina T, Pedrosa AL, Cruz AK. Leishmania major phosphoglycerate kinase transcript and protein stability contributes to differences in isoform expression levels. Exp Parasitol. 2015;159:222-6.
40. Mandal G, Mandal S, Sharma M, et al. Species-specific antimonial sensitivity in Leishmania is driven by post-transcriptional regulation of AQP1. PLoS Negl Trop Dis. 2015;9(2):e0003500.
2. Klatt S, Simpson L, Maslov DA, Konthur Z. Leishmania tarentolae: Taxonomic classification and its application as a promising biotechnological expression host. PLoS Negl Trop Dis. 2019;13(7):e0007424.
3. de Oliveira TA, Silva Wd, da Rocha Torres N, et al. Application of the LEXSY Leishmania tarentolae system as a recombinant protein expression platform: A review. Process Biochemistry. 2019;87:164-73.
4. Breitling R. The LEXSY platform for recombinant protein expression. Farm animal proteomics Wageningen Academic Publishers. 2013.
5. Dortay H, Mueller-Roeber B. A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker. Microb Cell Fact. 2010;9:29.
6. Kazemi, B, Moazzen F, Abadi A, Ghadjari A. Isolation a lizard Leishmania promastigote from its natural hostin Iran. Journal of Biological Sciences. 2004;4(5):620-623
7. Abdi Ghavidel A, Aghamiri S, Jajarmi V, Bandehpour M, Kazemi B. The Influence of Different Culture Media on the Growth and Recombinant Protein Production of Iranian Lizard Leishmania Promastigote. Iran J Parasitol. 2022;17(4):543-53.
8. Ehya F, Kalantari S, Bandehpour M, Kazemi B. Preliminary Information of Iranian Lizard Leishmania Promastigote Transcriptome Sequencing by Next Generation Sequencing (NGS) Method. Iran J Parasitol. 2023;18(3):362-368.
9. Yadegari Z, Bandehpour M, Kazemi B, Sharifi-Sarasiabi K. Expression of Recombinant Human Amelogenin in Iranian Lizard Leishmania and Its Biological Function Assay. Iran J Public Health. 2015;44(7):987-96.
10. Taromchi AH, Kazemi B, Mahmazi S, Bandehpour M. Heterologous Expression of Human IL-29 (IFN-Î) in Iranian Lizard Leishmania. Iran J Biotechnol. 2013;11(3):168-74.
11. Donyavi T, Bandehpour M, Kazemi B. Preparation of transgenic Iranian lizard Leishmania coding HIL-12. Iran J Microbiol. 2017;9(5):305-11.
12. Huang LF, Sinaga DS, Tan CC, Hsieh SJM, Huang CH. Expression of Recombinant Human Octamer-Binding Transcription Factor 4 in Rice Suspension Cells. Int J Mol Sci. 2021;22(3):1409.
13. Kim NS, Kim TG, Kim OH, et al. Improvement of recombinant hGM-CSF production by suppression of cysteine proteinase gene expression using RNA interference in a transgenic rice culture. Plant Mol Biol. 2008;68:263-75.
14. Duwadi K, Chen L, Menassa R, Dhaubhadel S. Identification, Characteriz-ation and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production. PLoS One. 2015;10(7):e0130556.
15. Kazemirad E, Reisi Nafchi H, Latifi A, Raoofian R, Mohebali M, Hajjaran H. Comparison of Cysteine Protease B Gene Expression between Clinical Isolates of Leishmania tropica, Leishmania major and Leishmania infantum. J Med Microbiol Infect Dis. 2019;7(3):72-8.
16. Buxbaum LU, Denise H, Coombs GH, Alexander J, Mottram JC, Scott P. Cysteine Protease B of Leishmania mexicana Inhibits Host Th1 Responses and Protective Immunity 1. J Immunol. 2003;171(7):3711-7.
17. Scala A, Micale N, Piperno A, et al. Targeting of the Leishmania mexicana cysteine protease CPB2.8ΔCTE by decorated fused benzo thiophene scaffold. RSC Adv. 2016;6(36):30628-35.
18. Suhr ST, Ramachandran R, Fuller CL, Veldman MB, Byrd CA, Goldman D. Highly-restricted, cell-specific expression of the simian CMV-IE promoter in transgenic zebrafish with age and after heat shock. Gene Expr Patterns. 2009;9(1):54-64.
19. Mirzaahmadi S, Asaadi-Tehrani G, Bandehpour M, et al. Expression of recombinant human coagulation factor VII by the Lizard Leishmania expression system. J Biomed Biotechnol. 2011; 2011:873874.
20. Zhang WW, Lypaczewski P, Matlashewski G. Application of CRISPR/Cas9-Mediated Genome Editing in Leishmania. In: Michels PAM, Ginger ML, Zilberstein D, editors. Trypanosomatids: Methods and Protocols. New York, NY: Springer US; 2020. p.199-224.
21. Zhang WW, Matlashewski G, Singh U. Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in Leishmania. mSphere. 2019;4(4):e00408-19.
22. Denise H, McNeil K, Brooks DR, Alexander J, Coombs GH, Mottram JC. Expression of multiple CPB genes encoding cysteine proteases is required for Leishmania mexicana virulence in vivo. Infect Immun. 2003;71(6):3190-5.
23. Hide M, Bras-Gonçalves R, Bañuls AL. Specific cpb copies within the Leishmania donovani complex: evolutionary interpretations and potential clinical implications in humans. Parasitology. 2007; 134(Pt 3):379-89.
24. Cruz A, Beverley SM. Gene replacement in parasitic protozoa. Nature. 1990; 348(6297): 171-3.
25. Dean S, Sunter J, Wheeler RJ, Hodkinson I, Gluenz E, Gull K. A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open Biol. 2015;5(1):140197.
26. Zhang WW, Matlashewski G. CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani. mBio. 2015;6(4):e00861.
27. Negreira GH, Monsieurs P, Imamura H, et al. High throughput single-cell genome sequencing gives insights into the generation and evolution of mosaic aneuploidy in Leishmania donovani. Nucleic Acids Res. 2021;50(1):293-305.
28. Duncan SM, Jones NG, Mottram JC. Recent advances in Leishmania reverse genetics: Manipulating a manipulative parasite. Mol Biochem Parasitol. 2017;216:30-8.
29. Níttolo AG, Bañuelos CP, Saborit JI, Tekiel V, Sánchez DO, Levy GV. TbRRM1 knockdown produces abnormal cell morphology and apoptotic-like death in the bloodstream form of T. brucei. Mol Biochem Parasitol. 2018;224:1-5.
30. Huynh TT, Huynh VT, Harmon MA, Phillips MA. Gene Knockdown of γ-Glutamylcysteine Synthetase by RNAi in the Parasitic Protozoa Trypanosoma brucei Demonstrates That It Is an Essential Enzyme. J Biol Chem. 2003;278(41):39794-800.
31. Abdi Ghavidel A, Aghamiri S, Raee P, et al. Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Leishmania Strains. Acta Parasitol. 2024; 69(1):121-134.
32. Alves CR, Côrte-Real S, DeFreitas Rosa M, GiovanniDe-Simone S. Detection of cysteine-proteinases in Leishmania amazonensis promastigotes using a cross-reactive antiserum. FEMS Microbiol Lett. 2000;186(2):263-7.
33. Casgrain PA, Martel C, McMaster WR, Mottram JC, Olivier M, Descoteaux A. Cysteine Peptidase B Regulates Leishmania mexicana Virulence through the Modulation of GP63 Expression. PLoS Pathog. 2016;12(5):e1005658.
34. Raymond F, Boisvert S, Roy G, et al. Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species. Nucleic Acids Res. 2012;40(3):1131-47.
35. Kushnir S, Gase K, Breitling R, Alexandrov K. Development of an inducible protein expression system based on the protozoan host Leishmania tarentolae. Protein Expr Purif. 2005;42(1):37-46.
36. Maree JP, Patterton HG. The epigenome of Trypanosoma brucei: a regulatory interface to an unconventional transcriptional machine. Biochim Biophys Acta. 2014;1839(9):743-50.
37. Castro Neto AL, Brito A, Rezende AM, Magalhães FB, de Melo Neto OP. In silico characterization of multiple genes encoding the GP63 virulence protein from Leishmania braziliensis: identification of sources of variation and putative roles in immune evasion. BMC Genomics. 2019;20(1):118.
38. Bifeld E, Lorenzen S, Bartsch K, Vasquez JJ, Siegel TN, Clos J. Ribosome Profiling Reveals HSP90 Inhibitor Effects on Stage-Specific Protein Synthesis in Leishmania donovani. mSystems. 2018; 3(6):e00214-18.
39. Azevedo A, Toledo JS, Defina T, Pedrosa AL, Cruz AK. Leishmania major phosphoglycerate kinase transcript and protein stability contributes to differences in isoform expression levels. Exp Parasitol. 2015;159:222-6.
40. Mandal G, Mandal S, Sharma M, et al. Species-specific antimonial sensitivity in Leishmania is driven by post-transcriptional regulation of AQP1. PLoS Negl Trop Dis. 2015;9(2):e0003500.
| Files | ||
| Issue | Vol 19 No 2 (2024) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijpa.v19i2.15855 | |
| Keywords | ||
| Iranian lizard leishmania Cysteine protease B Recombinant human factor VII | ||
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How to Cite
1.
Abdi Ghavidel A, Bandehpour M, Noori E, Jajarmi V, Kazemi B. Comparing the Yield of Recombinant Human Factor VII Protein Expressed by the rDNA-Promoter with the CMV-Promoter in Iranian Lizard Leishmania. Iran J Parasitol. 2024;19(2):192-202.
