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

Piotr Skowron , Natalia Krawczun , Joanna Żebrowska , Daria Krefft , Olga Żołnierkiewicz , Marta Bielawa , Joanna Jeżewska-Frąckowiak , Łukasz Janus , Małgorzata Witkowska , Małgorzata Palczewska , Adriana Schumacher , Anna Wardowska , Milena Deptuła , Artur Czupryn , Piotr Mucha , Arkadiusz Piotrowski , Paweł Sachadyn , Sylwia Rodziewicz-Motowidło , Michał Pikuła , Agnieszka Żylicz-Stachula

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.
Author Piotr Skowron (FCh/DMBt/LGE)
Piotr Skowron,,
- Laboratory of Genetic Engineering
, Natalia Krawczun (FCh/DMBt)
Natalia Krawczun,,
- Department of Molecular Biotechnology
, Joanna Żebrowska (FCh/DMBt/LGE)
Joanna Żebrowska,,
- Laboratory of Genetic Engineering
, Daria Krefft (FCh/DMBt/LGE)
Daria Krefft,,
- Laboratory of Genetic Engineering
, Olga Żołnierkiewicz (FCh/DMBt/LGE)
Olga Żołnierkiewicz,,
- Laboratory of Genetic Engineering
, Marta Bielawa
Marta Bielawa,,
-
, Joanna Jeżewska-Frąckowiak (FCh/DMBt/LGE)
Joanna Jeżewska-Frąckowiak,,
- Laboratory of Genetic Engineering
, Łukasz Janus (FCh/DMBt/LGE)
Łukasz Janus,,
- Laboratory of Genetic Engineering
, Małgorzata Witkowska (FCh/DMBt/LGE)
Małgorzata Witkowska,,
- Laboratory of Genetic Engineering
, Małgorzata Palczewska (FCh/DMBt/LGE)
Małgorzata Palczewska,,
- Laboratory of Genetic Engineering
et al.`
Journal seriesMaterials Science & Engineering C - Materials for Biological Applications, [Materials Science and Engineering C], ISSN 0928-4931, e-ISSN 1873-0191, (N/A 140 pkt)
Issue year2020
Vol108
Pages1-15
Publication size in sheets0.70
Article number110426
Referenced article (e.g. original article) Skowron Piotr, Krawczun Natalia, Żebrowska Joanna [et al.]: Data regarding a new, vector-enzymatic DNA fragment amplification-expression technology for the construction of artificial, concatemeric DNA, RNA and proteins, as well as biological effects of selected polypeptides obtained using this method, in: Data in Brief, vol. 28, 2020, pp. 1-16, Article number:105069, DOI:10.1016/j.dib.2019.105069
ASJC Classification2210 Mechanical Engineering; 2211 Mechanics of Materials; 2500 General Materials Science; 3104 Condensed Matter Physics
DOIDOI:10.1016/j.msec.2019.110426
URL https://doi.org/10.1016/j.msec.2019.110426
Languageen angielski
Score (nominal)140
Score sourcejournalList
ScoreMinisterial score = 140.0, 05-06-2020, ArticleFromJournal
Publication indicators WoS Citations = 0.000; Scopus SNIP (Source Normalised Impact per Paper): 2017 = 1.384; WoS Impact Factor: 2018 = 4.959 (2) - 2018=4.708 (5)
Citation count*3 (2020-07-31)
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* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.
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