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

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 Sap I 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. • A break-through vector-enzymatic DNA fragment amplification-expression technology for construction of concatemeric DNA, RNA and proteins is presented. • Concatemers are ready for genetic expression and can contain hundreds of copies of monomers arranged in ordered, head-to-tail fasion. • The technology for construction of novel biomaterials has numerous applications: biomedical, industrial, research and environmental. • The technology has already being practically applied, is covered by issued patent no PL 228341-B1 as well as pending worldwide patent applications. [ABSTRACT FROM AUTHOR]