Volume 14, Issue 2 (2-2026)                   JoMMID 2026, 14(2): 149-158 | Back to browse issues page

Ethics code: This work does not contain any studies involving human or animal subjects.

XML Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Barkhordari F, Hosseini Shalmaei M, Naderi S, Raigani M, Kayalha Y, Bayat E, et al . The Influence of IF2 and SUMO Fusion Tags on the Expression and Solubility of the SpCas9 Protein. JoMMID 2026; 14 (2) :149-158
URL: http://jommid.pasteur.ac.ir/article-1-880-en.html
The Influence of IF2 and SUMO Fusion Tags on the Expression and Solubility of the SpCas9 Protein
Farzaneh Barkhordari , Mahshid Hosseini Shalmaei , Setayesh Naderi , Mozhgan Raigani , Yeganeh Kayalha , Elham Bayat , Negin Sadat Hashemi Aval , Ahmad Adeli , Fatemeh Davami *
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
Abstract:   (10 Views)
Introduction: The expression of the Cas9 protein in the prokaryotic host Escherichia coli is often associated with the formation of inclusion bodies. This study aimed to express and purify the Cas9 protein in large quantities and soluble form in E. coli BL21 (DE3) for use in the CRISPR/Cas9 system. Due to the large size of Cas9, two solubility-enhancing fusion partners with relatively low molecular weights, initiation factor 2 (IF2) and small ubiquitin-like modifier (SUMO), were utilized. Methods: The IF2 and SUMO gene sequences, 480 and 300 bp in length, respectively, were amplified by PCR and cloned into the pET-21a-3xNLS-SpCas9 vector. The expression of both recombinant proteins, with approximate molecular weights of 176 and 166 kDa, was confirmed by SDS-PAGE and Western blotting. Protein purification was performed using Ni-NTA affinity chromatography. Results: The presence of IF2 as a fusion partner increased the percentage of Cas9 detected in the supernatant from 5.51% to 6.99%. Moreover, under optimal cultivation conditions, the recombinant IF2-Cas9 protein was produced in greater quantities than Cas9 alone (18.50 versus 13.65 mg/L). The solubility analysis revealed that SUMO-Cas9 exhibited higher solubility (9.25%) than Cas9 alone (5.51%), representing a 67.9 ± 8.2% increase in soluble expressio. Conclusion: While both fusion partners enhanced Cas9 expression in E. coli, SUMO demonstrated superior solubility-enhancing capability compared to IF2; however, the functional activity of the purified protein remains to be confirmed.
Keywords: SpCas9 expression, CRISPR/Cas9, Initiation factor 2, Solubility-enhancing tag, Small ubiquitin-like modifier, Escherichia coli
Full-Text [PDF 1049 kb]   (3 Downloads)    
Type of Study: Original article | Subject: Other
Received: 2026/09/7 | Accepted: 2026/02/21 | Published: 2026/09/7
References
1. Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 2005; 60 (2): 174-82. [DOI:10.1007/s00239-004-0046-3] [PMID]
2. Gagnon JA, Valen E, Thyme SB, Huang P, Akhmetova L, Pauli A, et al. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS One. 2014; 9 (5): e98186. [DOI:10.1371/journal.pone.0098186] [PMID]
3. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014; 346 (6213): 1258096. [DOI:10.1126/science.1258096] [PMID]
4. Nishimasu H, Ran FA, Hsu PD, Konermann S, Arora S, Kocha JS, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014; 156 (5): 935-49. [DOI:10.1016/j.cell.2014.02.001] [PMID]
5. Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature. 2012; 482 (7385): 331-8. [DOI:10.1038/nature10886] [PMID]
6. Frangoul H, Altshuler D, Cappellini MD, Chen YS, Domm J, Eustace BK, et al. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N Engl J Med. 2021; 384 (3): 252-60. [DOI:10.1056/NEJMoa2031054] [PMID]
7. Joung J, Ladha A, Saito M, Kim NG, Woolley AE, Segel M, et al. Detection of SARS-CoV-2 with SHERLOCK one-pot testing. N Engl J Med. 2020; 383 (15): 1492-4. [DOI:10.1056/NEJMc2026172] [PMID]
8. Dash PK, Kaminski R, Bella R, Su H, Mathews S, Ahooyi TM, et al. Sequential LASER ART and CRISPR treatments eliminate HIV-1 in a subset of infected humanized mice. Nat Commun. 2019; 10 (1): 2753. [DOI:10.1038/s41467-019-10366-y] [PMID] [PMCID]
9. Kim S, Kim D, Cho SW, Kim J, Kim JS. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 2014; 24 (6): 1012-9. [DOI:10.1101/gr.171322.113] [PMID]
10. Vakulskas CA, Behlke MA. Evaluation and reduction of CRISPR off-target cleavage events. Nucleic Acid Ther. 2019; 29 (4): 167-74. [DOI:10.1089/nat.2019.0790] [PMID] [PMCID]
11. DeWitt MA, Corn JE, Carroll D. Genome editing via delivery of Cas9 ribonucleoprotein. Methods. 2017; 121-122: 9-15. [DOI:10.1016/j.ymeth.2017.04.003] [PMID] [PMCID]
12. Liang X, Potter J, Kumar S, Ravinder N, Chesnut JD. Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA. J Biotechnol. 2017; 241: 136-46. [DOI:10.1016/j.jbiotec.2016.11.011] [PMID]
13. Mout R, Ray M, Yesilbag Tonga G, Lee YW, Tay T, Sasaki K, et al. Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing. ACS Nano. 2017; 11 (3): 2452-8. [DOI:10.1021/acsnano.6b07600] [PMID]
14. Gundry MC, Brunetti L, Lin A, Mayle AE, Kitano A, Wagner D, et al. Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9. Cell Rep. 2016; 17 (5): 1453-61. [DOI:10.1016/j.celrep.2016.09.092] [PMID] [PMCID]
15. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337 (6096): 816-21. [DOI:10.1126/science.1225829] [PMID]
16. Liu C, Zhang L, Liu H, Cheng K. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. J Control Release. 2017; 266: 17-26. [DOI:10.1016/j.jconrel.2017.09.012] [PMID] [PMCID]
17. Sørensen HP, Sperling-Petersen HU, Mortensen KK. A favorable solubility partner for the recombinant expression of streptavidin. Protein Expr Purif. 2003; 32 (2): 252-9. [DOI:10.1016/j.pep.2003.07.001] [PMID]
18. Dyson MR, Shadbolt SP, Vincent KJ, Perera RL, McCafferty J. Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC Biotechnol. 2004; 4: 32. [DOI:10.1186/1472-6750-4-32] [PMID] [PMCID]
19. Klock HE, Koesema EJ, Knuth MW, Lesley SA. Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins. 2008; 71 (2): 982-94. [DOI:10.1002/prot.21786] [PMID]
20. Yang WC, Welsh JP, Lee J, Cooke JP, Swartz JR. Solubility partner IF2 domain I enables high yield synthesis of transducible transcription factors in Escherichia coli. Protein Expr Purif. 2011; 80 (1): 145-51. [DOI:10.1016/j.pep.2011.06.017] [PMID] [PMCID]
21. Sati SP, Singh SK, Kumar N, Sharma A. Extra terminal residues have a profound effect on the folding and solubility of a Plasmodium falciparum sexual stage-specific protein over-expressed in Escherichia coli. Eur J Biochem. 2002; 269 (21): 5259-63. [DOI:10.1046/j.1432-1033.2002.03237.x] [PMID]
22. Malhotra A. Tagging for protein expression. Methods Enzymol. 2009; 463: 239-58. [DOI:10.1016/S0076-6879(09)63016-0] [PMID]
23. Moreno JM, Dyrskjøt L, Kristensen JE, Mortensen KK, Sperling-Petersen HU. Characterization of the domains of Escherichia coli initiation factor IF2 responsible for recognition of the ribosome. FEBS Lett. 1999; 455 (1-2): 130-4. [DOI:10.1016/S0014-5793(99)00858-3] [PMID]
24. Butt TR, Edavettal SC, Hall JP, Mattern MR. SUMO fusion technology for difficult-to-express proteins. Protein Expr Purif. 2005; 43 (1): 1-9. [DOI:10.1016/j.pep.2005.03.016] [PMID] [PMCID]
25. Butt TR, Jonnalagadda S, Monia BP, Sternberg EJ, Marsh JA, Stadel JM, et al. Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli. Proc Natl Acad Sci U S A. 1989; 86 (8): 2540-4. [DOI:10.1073/pnas.86.8.2540] [PMID] [PMCID]
26. Wittliff JL, Wenz LL, Dong J, Nawaz Z, Butt TR. Expression and characterization of an active human estrogen receptor as a ubiquitin fusion protein from Escherichia coli. J Biol Chem. 1990; 265 (35): 22016-22. [DOI:10.1016/S0021-9258(18)45841-3] [PMID]
27. Wang Z, Li H, Guan W, Ling H, Wang Z, Mu T, et al. Human SUMO fusion systems enhance protein expression and solubility. Protein Expr Purif. 2010; 73 (2): 203-8. [DOI:10.1016/j.pep.2010.05.001] [PMID]
28. Zuo X, Li S, Hall J, Mattern MR, Tran H, Shoo J, et al. Enhanced expression and purification of membrane proteins by SUMO fusion in Escherichia coli. J Struct Funct Genomics. 2005; 6 (2-3): 103-11. [DOI:10.1007/s10969-005-2664-4] [PMID] [PMCID]
29. Zuo X, Mattern MR, Tan R, Li S, Hall J, Sterner DE, et al. Expression and purification of SARS coronavirus proteins using SUMO-fusions. Protein Expr Purif. 2005; 42 (1): 100-10. [DOI:10.1016/j.pep.2005.02.004] [PMID] [PMCID]
30. Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing. Theranostics. 2021; 11 (2): 614-48. [DOI:10.7150/thno.47007] [PMID]
31. Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014; 5: 172. [DOI:10.3389/fmicb.2014.00172]
32. Peleg Y, Unger T. Resolving bottlenecks for recombinant protein expression in E. coli. Methods Mol Biol. 2012; 800: 173-86. [DOI:10.1007/978-1-61779-349-3_12] [PMID]
33. Francis DM, Page R. Strategies to optimize protein expression in E. coli. Curr Protoc Protein Sci. 2010; Chapter 5: Unit 5.24.
34. Ahmad I, Nawaz N, Darwesh NM, Ur Rahman S, Mustafa MZ, Khan SB, et al. Overcoming challenges for amplified expression of recombinant proteins using Escherichia coli. Protein Expr Purif. 2018; 144: 12-8. [DOI:10.1016/j.pep.2017.11.005] [PMID]
35. Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo X, Butt TR. Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci. 2006;15 (1): 182-9. [DOI:10.1110/ps.051812706] [PMID] [PMCID]
36. Aslam M, Gull I, Abbas Z, Athar M. Soluble expression of IFNα2-Tα1 fusion protein in Escherichia coli by N-terminal SUMO fusion and its anti-proliferative activity. Pak J Zool. 2018; 50 (6): 2291-8. [DOI:10.17582/journal.pjz/2018.50.4.1413.1419]
37. Bi J, Tiong E, Koo YS, Zhou W, Wong FT. Further characterization and engineering of an 11-amino acid motif for enhancing recombinant soluble protein expression. Microb Cell Fact. 2025; 24 (1): 122. [DOI:10.1186/s12934-025-02738-5] [PMID] [PMCID]
38. Arakawa T, Akuta T. Beyond purification: evolving roles of fusion tags in biotechnology. Curr Issues Mol Biol. 2025; 47 (9): 768. [DOI:10.3390/cimb47090768] [PMID] [PMCID]
Add your comments about this article : Your username or Email:
CAPTCHA
Send email to the article author

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.