Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins
| Autor: | Renaud Vincentelli, Carlos M. G. A. Fontes, Ana Filipa Sequeira, Joana L. A. Brás, Catarina I. P. D. Guerreiro |
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| Přispěvatelé: | Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), European Project: 278346,EC:FP7:HEALTH,FP7-HEALTH-2011-two-stage,VENOMICS(2011), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA) |
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Assembly PCR Computational biology Biology Gene synthesis Polymerase cycling assembly Protein Engineering Venom peptides 03 medical and health sciences Escherichia coli Genes Synthetic Animals Gene Genetics Expression vector 030102 biochemistry & molecular biology [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry Molecular Biology/Structural Biology [q-bio.BM] Oligonucleotide Drug discovery Methodology Article Ligation-independent cloning Snakes Protein engineering Gene design [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM] Recombinant Proteins High-Throughput Screening Assays Molecular Weight [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry Molecular Biology/Biomolecules [q-bio.BM] 030104 developmental biology Batch Cell Culture Techniques Codon usage bias Algorithms Biotechnology Snake Venoms |
| Zdroj: | BMC Biotechnology BMC Biotechnology, BioMed Central, 2016, 16 (1), ⟨10.1186/s12896-016-0316-3⟩ BMC Biotechnology, 2016, 16 (1), ⟨10.1186/s12896-016-0316-3⟩ |
| ISSN: | 1472-6750 |
| DOI: | 10.1186/s12896-016-0316-3⟩ |
| Popis: | Background Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression. Results Here, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins. Conclusions An automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0316-3) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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