Skip to main content
SpringerLink
Log in

Nuclear localization signal peptides enhance genetic transformation of Dunaliella salina

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Background

Dunaliella salina (D. salina) expression system shows a very attractive application prospect, but it currently has a technical bottleneck, namely the low or unstable expression of recombinant proteins. Given the characteristics of cell-penetrating peptides or/and nuclear localization signal (NLS) peptides, this study is the first attempt to improve the transformation rate of foreign gene with trans-activating transcriptional (TAT) protein or/and NLS peptides.

Methods and results

Using salt gradient method, exogenous plasmids were transferred into D. salina cells with TAT or TAT/NLS complexes simultaneously. The β-glucuronidase gene expression was identified by means of histochemical stain and RT-qPCR detection. Through observation with light microscope, TAT-mediating cells exhibit an apparent cytotoxicity even at ratios of 0.5, no significant toxicity was noted in the TAT/plasmid/NLS complex group. It is obvious that with the addition of peptides the toxicity decreases significantly. Histochemical staining showed that the transformants presented blue color under light microscope, but the negative control and blank control are not. Furthermore, based on a TAT/plasmids ratio of 4 with 10 µg NLS peptides mediation, RT-qPCR results demonstrated that the transcripts of target gene were increased by 269 times than that of control group.

Conclusions

This study demonstrated that combination of TAT and NLS peptides can significantly improve the transformation rate and expression level of foreign gene in D. salina system. It offers a promising way for promoting the application and development of D. salina bioreactor.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Data availability

The data of this article is included within the article. And, the data and materials can also be requested from the corresponding author and the first author.

Abbreviations

D. salina :

Dunaliella salina

CPPs:

Cell-penetrating peptides

TAT:

Trans-activating protein

NLS:

Nuclear localization signal

GUS:

Beta-glucuronidase

RT-qPCR:

Real time fluorescent quantitative PCR

RNP:

Ribonucleoprotein

References

  1. Feng SY, Li XB, Xu ZS, Qi JJ (2014) Dunaliella salina as a novel host for the production of recombinant proteins. Appl Microbiol Biot 98(10):4293–4300. https://doi.org/10.1007/s00253-014-5636-4

    Article  CAS  Google Scholar 

  2. Feng SY, Feng WP, Zhao L, Gu HH, Li QH, Shi K, Guo SX, Zhang NN (2014) Preparation of transgenic Dunaliella salina for immunization against white spot syndrome virus in crayfish. Arch Virol 159(3):519–525. https://doi.org/10.1007/s00705-013-1856-7

    Article  CAS  Google Scholar 

  3. Chai XJ, Chen HX, Xu WQ, Xu YW (2013) Expression of soybean kunitz trypsin inhibitor gene SKTI in Dunaliella salina. J Appl Phycol 25(1):139–144. https://doi.org/10.1007/s10811-012-9847-8

    Article  CAS  Google Scholar 

  4. Castellanos-Huerta I, Gómez-Verduzco G, Tellez-Isaias G, Ayora-Talavera G, Bañuelos-Hernández B, Petrone-García VM, Velázquez-Juárez G, Fernández-Siurob I (2022) Transformation of Dunaliella salina by Agrobacterium tumefaciens for the expression of the hemagglutinin of avian influenza virus H5. Microorganisms 10(2):361. https://doi.org/10.3390/microorganisms10020361

    Article  CAS  Google Scholar 

  5. Poungpair O, Bangphoomi K, Chaowalit P, Sawasdee N, Saokaew N, Choowongkomon K, Chaicumpa W, Yenchitsomanus PT (2014) Generation of human single-chain variable fragment antibodies specific to dengue virus non-structural protein 1 that interfere with the virus infectious cycle. MAbs 6(2):474–482. https://doi.org/10.4161/mabs.27874

    Article  Google Scholar 

  6. Song GN, Wang W, Hu LN, Liu Y, Feng SY (2019) An exploration of the rapid transformation method for Dunaliella salina system. AMB Express 9(1):181. https://doi.org/10.1186/s13568-019-0905-3

    Article  CAS  Google Scholar 

  7. Peraro L, Kritzer JA (2018) Emerging methods and design principles for cell-penetrant peptides. Angew Chem Int Edit 57(37):11868–11881. https://doi.org/10.1002/anie.201801361

    Article  CAS  Google Scholar 

  8. Thagun C, Chuah J, Numata K (2019) Targeted gene delivery: targeted gene delivery into various plastids mediated by clustered cell-penetrating and chloroplast-targeting peptides. Adv Sci 6(23):1902064. https://doi.org/10.1002/advs.201970142

    Article  CAS  Google Scholar 

  9. Wu Y, Yao X, Chen Y, Li YP, Tian WQ (2017) Advance of DNA and CCPs-based nanocarriers in drug delivery systems. Bio-Med Mater Eng 28(s1):S255–S261. https://doi.org/10.3233/bme-171648

    Article  Google Scholar 

  10. Fu T, Kuo P, Lu Y, Lin H, Chang M (2020) Cell penetrating peptide as a high safety anti-inflammation ingredient for cosmetic applications. Biomolecules 10(1):1011

    Article  Google Scholar 

  11. Ruseska I, Zimmer A (2020) Internalization mechanisms of cell-penetrating peptides. Beilstein J Nanotechnol 11:101–123. https://doi.org/10.1002/9783527626830.ch7

    Article  CAS  Google Scholar 

  12. Liu J, Afshar S (2020) In vitro assays: friends or foes of cell-penetrating peptides. Int J Mol Sci 21(13):4719. https://doi.org/10.3390/ijms21134719

    Article  CAS  Google Scholar 

  13. Kardani K, Bolhassani A (2021) Cppsite 2.0: an available database of experimentally validated cell-penetrating peptides predicting their secondary and tertiary structures. J Mol Biol 433(11):166703. https://doi.org/10.1016/j.jmb.2020.11.002

    Article  CAS  Google Scholar 

  14. Klabenkova K, Fokina A, Stetsenko D (2021) Chemistry of peptide-oligonucleotide conjugates: a review. Molecules 26(17):5420. https://doi.org/10.3390/molecules26175420

    Article  CAS  Google Scholar 

  15. Sakamoto K, Morishita T, Aburai K, Sakai K, Abe M, Nakase I, Futaki S, Sakai H (2020) Key process and factors controlling the direct translocation of cell-penetrating peptide through bio-membrane. Int J Mol Sci 21(15):5466. https://doi.org/10.3390/ijms21155466

    Article  CAS  Google Scholar 

  16. Cokol M, Nair R, Rost B (2000) Finding nuclear localization signals. EMBO Rep 1(5):411–415. https://doi.org/10.1093/embo-reports/kvd092

    Article  CAS  Google Scholar 

  17. Soniat M, Chook Y (2015) Nuclear localization signals for four distinct karyopherin-β nuclear import systems. Biochem J 468(3):353–362. https://doi.org/10.1042/bj20150368

    Article  CAS  Google Scholar 

  18. Bogacheva M, Egorova A, Slita A, Maretina M, Baranov V, Kiselev A (2017) Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery. Bioorg Med Chem Lett 27(21):4781–4785. https://doi.org/10.1016/j.bmcl.2017.10.001

    Article  CAS  Google Scholar 

  19. Fernandez J, Machado AK, Lyonnais S, Chamontin C, Gärtner K, Léger T, Henriquet C, Garcia C, Portilho DM, Pugnière M, Chaloin L, Muriaux D, Yamauchi Y, Blaise M, Nisole S, Arhel NJ (2019) Transportin-1 binds to the HIV-1 capsid via a nuclear localization signal and triggers uncoating. Nat Microbiol 4(11):1840–1850. https://doi.org/10.1038/s41564-019-0575-6

    Article  CAS  Google Scholar 

  20. Owji H, Nezafat N, Negahdaripour M, Hajiebrahimi A, Ghasemi Y (2018) A comprehensive review of signal peptides: structure, roles, and applications. Eur J Cell Biol 97(6):422–441. https://doi.org/10.1016/j.ejcb.2018.06.003

    Article  CAS  Google Scholar 

  21. Yi WJ, Yang J, Li C, Wang HY, Liu CW, Tao L, Cheng SX, Cargo Rx Z, Zhang XZ (2011) Enhanced nuclear import and transfection efficiency of TAT peptide-based gene delivery systems modified by additional nuclear localization signals. Bioconjugate Chem 23(1):125–134. https://doi.org/10.1021/bc2005472

    Article  CAS  Google Scholar 

  22. Kanazawa T, Yamazaki M, Fukuda T, Takashima Y, Okada H (2015) Versatile nuclear localization signal-based oligopeptide as a gene vector. Biol Pharm Bull 38(4):559–565. https://doi.org/10.1248/bpb.b14-00706

    Article  CAS  Google Scholar 

  23. Hyman JM, Geihe EI, Trantow BM, Parvin B, Wender PA (2012) A molecular method for the delivery of small molecules and proteins across the cell wall of algae using molecular transporters. Proc Natl Acad Sci USA 109(33):13225–13230. https://doi.org/10.1073/pnas.1202509109

    Article  Google Scholar 

  24. Suresh A, Kim Y (2013) Translocation of cell penetrating peptides on Chlamydomonas reinhardtii. Biotechnol Bioeng 110(10):2795–2801. https://doi.org/10.1002/bit.24935

    Article  CAS  Google Scholar 

  25. Wei Y, Niu J, Li H, Huang A, He L, Wang G (2015) Cell penetrating peptide can transport dsRNA into microalgae with thin cell walls. Algal Res 8:135–139. https://doi.org/10.1016/j.algal.2015.02.002

    Article  Google Scholar 

  26. Gadamchetty P, Mullapudi P, Sanagala R, Markandan M, Polumetla AK (2019) Genetic transformation of Chlorella vulgaris mediated by HIV-TAT peptide. 3 Biotech 9(4):139. https://doi.org/10.1007/s13205-019-1671-2

    Article  Google Scholar 

  27. Kang S, Suresh A, Kim YC (2017) A highly efficient cell penetrating peptide pVEC-mediated protein delivery system into microalgae. Algal Res 24:360–367. https://doi.org/10.1016/j.algal.2017.04.022

    Article  Google Scholar 

  28. Feng SY, Xue LX, Liu H, Lu P (2009) Improvement of efficiency of genetic transformation for Dunaliella salina by glass beads method. Mol Biol Rep 36(6):1433–1439. https://doi.org/10.1007/s11033-008-9333-1

    Article  CAS  Google Scholar 

  29. Sun Y, Yang ZY, Gao XS, Li QY, Zhang QQ, Xu ZK (2005) Expression of foreign genes in Dunaliella by electroporation. Mol Biotechnol 30(3):185–192. https://doi.org/10.1385/mb:30:3:185

    Article  CAS  Google Scholar 

  30. Tan CP, Qin S, Zhang Q, Jiang P, Zhao FQ (2005) Establishment of a micro-particle bombardment transformation system for Dunaliella salina. J Microbiol 43(4):361–365. https://doi.org/10.1128/CMR.00001-08

    Article  CAS  Google Scholar 

  31. Feng SY, Hu LN, Zhang QH, Zhang FQ, Liu Y (2020) CRISPR/Cas technology promotes the various application of Dunaliella salina system. Appl Microbiol Biot 104(20):8621–8630. https://doi.org/10.1007/s00253-020-10892-6

    Article  CAS  Google Scholar 

  32. Hu LN, Feng SY, Liang GF, Du JX, Niu CL (2021) CRISPR/Cas9-induced β-carotene hydroxylase mutation in Dunaliella salina CCAP19/18. AMB Express 11(1):83

    Article  CAS  Google Scholar 

  33. Xu Y, Liang W, Qiu Y, Cespi M, Palmieri GF, Mason AJ, Lam JKW (2016) Incorporation of a nuclear localization signal in pH responsive LAH4-L1 peptide enhances transfection and nuclear uptake of plasmid DNA. Mol Pharm 13(9):3141–3152. https://doi.org/10.1021/acs.molpharmaceut.6b00338

    Article  CAS  Google Scholar 

Download references

Funding

This study was founded by the National Natural Science Foundation of China (No. U1804112), and the Zhong Jing Core Scholar’s Research Initial Fund of Henan University of Chinese Medicine (No. 00104311-2021).

Author information

Author notes

  1. Shuying Feng and Lina Hu contributed equally to this work.

Authors and Affiliations

  1. Medical College, Henan University of Chinese Medicine, No. 156, Jinshui East Road, Zhengzhou, 450046, China

    Shuying Feng, Lina Hu, Aifang Li, Shuxuan Li, Yalan Li, Chunling Niu, Baiyan Wang & Sugai Yin

  2. School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou, 450046, Henan, China

    Shuying Feng & Tao Guo

Authors
  1. Shuying Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lina Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aifang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shuxuan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yalan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chunling Niu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Baiyan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sugai Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Tao Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

LN, SY wrote the manuscript. LN, SY, AF drew the pictures. CL, BY, YL, SX, YX conduct RT-qPCR and histochemical staining analysis. T polished the paper. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Shuying Feng or Tao Guo.

Ethics declarations

Competing interest

The authors declare no conflicts of interest in relation to this research and its publication.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Feng, S., Hu, L., Li, A. et al. Nuclear localization signal peptides enhance genetic transformation of Dunaliella salina. Mol Biol Rep 50, 1459–1467 (2023). https://doi.org/10.1007/s11033-022-08159-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-022-08159-6

Keywords

Search

Navigation