Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxinsReport as inadecuate




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BMC Biotechnology

, 16:86

First Online: 01 December 2016Received: 16 April 2016Accepted: 23 November 2016

Abstract

BackgroundGene 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.

ResultsHere, 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.

ConclusionsAn 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.

KeywordsGene synthesis Assembly PCR Gene design Venom peptides AbbreviationsCAICodon Adaptation Index

DA-PCRDual asymmetrical PCR

GCGuanine-cytosine

IPSImproved PCR synthesis

KbKilobase

LICLigation-Independent Cloning protocol

NtNucleotides

OE-PCROverlap-extension PCR

PASPCR-based accurate synthesis

PCAPolymerase chain assembly

PCA-DTPolymerase chain assembly using DNA template

PCA-DTFPolymerase chain assembly DNA template-free

PTDSPCR-based two-step DNA synthesis

SGSSimplified gene synthesis

TBIOThermodynamically balanced inside-out technology

TEVTobacco Etch Virus protease.

Electronic supplementary materialThe online version of this article doi:10.1186-s12896-016-0316-3 contains supplementary material, which is available to authorized users.

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Author: Ana Filipa Sequeira - Joana L. A. Brás - Catarina I. P. D. Guerreiro - Renaud Vincentelli - Carlos M. G. A. Fontes

Source: https://link.springer.com/







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