An efficient method for the construction of artificial, concatemeric DNA, RNA and proteins with genetically programmed functions, using a novel, vector-enzymatic DNA fragment amplification-expression technology

Abstract

De novo designed bioactive molecules, such as DNA, RNA and peptides, are utilized in increasingly diverse scientific, industrial and biomedical applications. Concatemerization of designed DNA, RNA and peptides may improve their stability, bioactivity and allow for gradual release of the bioactive molecule at the intended destination. In this context, we developed a new method enabling the formation of DNA concatemers for the production of artificial, repetitive genes, encoding concatemeric RNAs and proteins of any nucleotide and amino-acid sequence. The technology recruits the Type IIS SapI restriction endonuclease (REase) for assembling DNA fragments in an ordered head-to-tail-orientation. Alternatively, other commercially available SapI isoschizomers can be used: LguI and thermostable BspQI. Four series of DNA vectors dedicated to the expression of newly formed, concatemeric open reading frames (ORFs), were designed and constructed to meet the technology needs. • Vector-enzymatic DNA fragment amplification technology. • Construction of DNA concatemers many times longer than those available with the use of current de novo gene synthesis methods. • Biosynthesis of protein tandem repeats with programmable function never seen in nature.

Development of hybrid biomicroparticles: cellulose exposing functionalized fusion proteins

“…Such a secretion leader arrangement enables expressed proteins to be secreted into the periplasmic space. Additionally, the MalE leader in general tends to improve the expression level of recombinant genes, which also applies to concatemeric genes, as we have shown previously [ 1 , 58 , 59 ].…”

“…In this study, a unique DNA FACE™ technology developed in our laboratory [ 1 , 57 – 59 ] for the construction of concatemeric proteins was used. This is an Escherichia coli -based DNA amplification-expression method allowing for the automatic assembly of concatemeric Open Reading Frames (ORFs) and proteins, based on the intertwined use of specialised vectors, containing universal DNA amplification modules, transcription/translation modules and two enzymes: SapI Type IIS restriction endonuclease, and T4 DNA ligase.…”