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Martinelli C, Colombo E, Piccini D, et al. An intrabody specific for the nucleophosmin carboxy-terminal mutant and fused to a nuclear localization sequence binds its antigen but fails to relocate it in the nucleus. Biotechnol Rep (Amst). 2014;3:27-33doi: 10.1016/j.btre.2014.05.008.
Martinelli, C., Colombo, E., Piccini, D., Sironi, C., Pelicci, P. G., & de Marco, A. (2014). An intrabody specific for the nucleophosmin carboxy-terminal mutant and fused to a nuclear localization sequence binds its antigen but fails to relocate it in the nucleus. Biotechnology reports (Amsterdam, Netherlands), 327-33. https://doi.org/10.1016/j.btre.2014.05.008
Martinelli, Chiara, et al. "An intrabody specific for the nucleophosmin carboxy-terminal mutant and fused to a nuclear localization sequence binds its antigen but fails to relocate it in the nucleus." Biotechnology reports (Amsterdam, Netherlands) vol. 3 (2014): 27-33. doi: https://doi.org/10.1016/j.btre.2014.05.008
Martinelli C, Colombo E, Piccini D, Sironi C, Pelicci PG, de Marco A. An intrabody specific for the nucleophosmin carboxy-terminal mutant and fused to a nuclear localization sequence binds its antigen but fails to relocate it in the nucleus. Biotechnol Rep (Amst). 2014 May 27;3:27-33. doi: 10.1016/j.btre.2014.05.008. eCollection 2014 Sep. PMID: 28626645; PMCID: PMC5466097.
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Biotechnol Rep (Amst). 2014 May 27;3:27-33. doi: 10.1016/j.btre.2014.05.008. eCollection 2014 Sep.

An intrabody specific for the nucleophosmin carboxy-terminal mutant and fused to a nuclear localization sequence binds its antigen but fails to relocate it in the nucleus.

Biotechnology reports (Amsterdam, Netherlands)

Chiara Martinelli, Emanuela Colombo, Daniele Piccini, Cristina Sironi, Pier Giuseppe Pelicci, Ario de Marco

Affiliations

  1. IFOM-IEO Campus, Via Adamello 16, 20139 Milan, Italy.
  2. Department of Experimental Oncology, IEO, Via Adamello 16, 20139 Milan, Italy.
  3. Department of Health Sciences, University of Milan, 20133 Milan, Italy.
  4. Department of Biomedical Sciences and Engineering, University of Nova Gorica, Glavni Trg 9, SI-5261 Vipava, Slovenia.

PMID: 28626645 PMCID: PMC5466097 DOI: 10.1016/j.btre.2014.05.008

Abstract

The cytoplasmic accumulation of NPM1 (NPMc+) is found in acute myeloid leukemia (AML) with NPM1 mutation. NPM1 must shuttle between nucleus and cytoplasm to assure physiological protein synthesis and, therefore, the elimination of NPMc+ is not a suitable therapeutic option. We isolated, characterized, and produced a functional scFv intrabody fused to nuclear localization signal(s) (NLS) that does not recognize NPM1 but binds to the mutant-specific C-terminal NES (nuclear export signal) of NPMc+, responsible for its cytoplasmic accumulation. The scFv-NLS fusion accumulated in the nuclei of wild type cells and strongly bound to its antigen in the cytoplasm of NPMc+ expressing cells. However, it failed to relocate the majority of NPMc+ in the nucleus, even when fused to four NLS. Our results show the technical feasibility of producing recombinant intrabodies with defined sub-cellular targeting and nuclear accumulation but the lack of information concerning the features that confer variable strength to the signal peptides impairs the development of biomolecules able to counteract pathological sub-cellular distribution of shuttling proteins.

Keywords: AML, acute myeloid leukemia; CRM1, Chromosomal Region Maintenance 1; GST, glutathione S-transferase; Intrabody; Leukemia; MBP, maltose binding protein; NES, nuclear export signal; NLS, nuclear localization signal; NPM1, nucleophosmin; NPMc+, cytoplasmic nucleophosmin; Protein shuttling; Recombinant fusion protein; Single-chain antibody; scFv, single-chain variable fragment

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