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Mater Sci Eng C Mater Biol Appl. 2020 Mar;108:110426. doi: 10.1016/j.msec.2019.110426. Epub 2019 Nov 13.

A vector-enzymatic DNA fragment amplification-expression technology for construction of artificial, concatemeric DNA, RNA and proteins for novel biomaterials, biomedical and industrial applications.

Author information

1
Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland; BioVentures Institute Ltd., Poznan 60-141, Poland. Electronic address: piotr.skowron@ug.edu.pl.
2
Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland; BioVentures Institute Ltd., Poznan 60-141, Poland.
3
Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland.
4
BioVentures Institute Ltd., Poznan 60-141, Poland.
5
Department of Embryology, Faculty of Medicine, Medical University of Gdansk, Gdansk 80-211, Poland; Department of Clinical Immunology and Transplantology, Faculty of Medicine, Medical University of Gdansk, 80-210, Poland.
6
Department of Clinical Immunology and Transplantology, Faculty of Medicine, Medical University of Gdansk, 80-210, Poland; Laboratory of Tissue Engineering and Regenerative Medicine, Department of Embryology, Faculty of Medicine, Medical University of Gdansk, Gdansk 80-211, Poland.
7
Laboratory of Tissue Engineering and Regenerative Medicine, Department of Embryology, Faculty of Medicine, Medical University of Gdansk, Gdansk 80-211, Poland.
8
Nencki Institute of Experimental Biology, Warsaw 02-093, Poland.
9
Department of Molecular Biochemistry, Faculty of Chemistry, University of Gdansk, 80-308, Poland.
10
Department of Biology and Pharmaceutical Botany, Faculty of Pharmacy, Medical University of Gdansk, 80-416, Poland; International Research Agenda - 3P Medicine Lab, Medical University of Gdansk, 80-416, Poland.
11
Department of Molecular Biotechnology and Microbiology, Faculty of Chemistry, Gdansk University of Technology, Gdansk 80-233, Poland.
12
Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Gdansk 80-308, Poland.

Abstract

A DNA fragment amplification/expression technology for the production of new generation biomaterials for scientific, industrial and biomedical applications is described. The technology enables the formation of artificial Open Reading Frames (ORFs) encoding concatemeric RNAs and proteins. It recruits the Type IIS SapI restriction endonuclease (REase) for an assembling of DNA fragments in an ordered head-to-tail-orientation. The technology employs a vector-enzymatic system, dedicated to the expression of newly formed, concatemeric ORFs from strong promoters. Four vector series were constructed to suit specialised needs. As a proof of concept, a model amplification of a 7-amino acid (aa) epitope from the S protein of HBV virus was performed, resulting in 500 copies of the epitope-coding DNA segment, consecutively linked and expressed in Escherichia coli (E. coli). Furthermore, a peptide with potential pro-regenerative properties (derived from an angiopoietin-related growth factor) was designed. Its aa sequence was back-translated, codon usage optimized and synthesized as a continuous ORF 10-mer. The 10-mer was cloned into the amplification vector, enabling the N-terminal fusion and multiplication of the encoded protein with MalE signal sequence. The obtained genes were expressed, and the proteins were purified. Conclusively, we show that the proteins are neither cytotoxic nor immunogenic and they have a very low allergic potential.

PMID:
31923928
DOI:
10.1016/j.msec.2019.110426

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