Abstract:Background: Numerous bacterial human growth hormone (hGH) expression methods under conventional fermentation and induction conditions have been described. Despite significant progress made in this area over the past several years, production of recombinant hGH by using cellular expression systems still requires further optimization. Fusion of the ubiquitin (Ub) tag to the hGH protein allowed to increase of the overall efficiency of the biosynthesis and improve the protein stability. Ub is a protein composed of… Show more
“…DNA sequence of pET28AMP_SapI-Ubq vector is provided in Supplemental data. The ubiquitin domain can be easily removed from a fusion protein by deubiquitinating proteases [6] . Fig.…”
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.
“…DNA sequence of pET28AMP_SapI-Ubq vector is provided in Supplemental data. The ubiquitin domain can be easily removed from a fusion protein by deubiquitinating proteases [6] . Fig.…”
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.
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