Engineering nuclear localization signals in modular protein vehicles for gene therapy.
Amino acids from 126 to 135 of the SV40 virus T antigen act as efficient nuclear localization signal during infection but also when fused to recombinant proteins. This peptide has been inserted into two alternative acceptor sites of a modified Escherichia coli beta-galactosidase which also displays a DNA-binding domain, a cell-binding motif for integrin alpha(v)beta(3) targeting and cell internalization, and a cryptic nuclear targeting signal naturally present in the bacterial enzyme. In cultured cells, the presence of the SV40 peptide enhances the expression of a delivered DNA up to 30-fold. However, the DNA expression levels are largely depending on the chosen insertion site for the SV40 segment concomitant to the structural impact of peptide accommodation on the protein vehicle. The structural stability of the hybrid protein, apparently critical for efficient gene transfer, is discussed in the context of modular protein engineering to develop non-viral vectors for gene therapy.
Institut de Biotecnologia i de Biomedicina and Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.
Anna
A
Arís
Antonio
A
Villaverde
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D019913:Nuclear Localization Signals; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D001426:Bacterial Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D004247:DNA; D001616:beta-Galactosidase