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- Development of a novel platform for high-throughput gene design and artificial gene synthesis to produce large libraries of recombinant venom peptides for drug discoveryPublication . Sequeira, Ana Filipa Pereira; Fontes, Carlos Mendes Godinho de Andrade; Vincentelli, Renaud; Guerreiro, Catarina Isabel Proença DuarteAnimal venoms are complex mixtures of biologically active molecules that, while presenting low immunogenicity, target with high selectivity and efficacy a variety of membrane receptors. It is believed that animal venoms comprise a natural library of more than 40 million different natural compounds that have been continuously fine-tuned during the evolutionary process to disturb cellular function. Within animal venoms, reticulated peptides are the most attractive class of molecules for drug discovery. However, the use of animal venoms to develop novel pharmacological compounds is still hampered by difficulties in obtaining these low molecular mass cysteine-rich polypeptides in sufficient amounts. Here, a high-throughput gene synthesis platform was developed to produce synthetic genes encoding venom peptides. The final goal of this project is the production of large libraries of recombinant venom peptides that can be screened for drug discovery. A robust and efficient Polymerase Chain Reaction (PCR) methodology was refined to assemble overlapping oligonucleotides into small artificial genes (< 500 bp) with high-fidelity. In addition, two bioinformatics tools were constructed to design multiple optimized genes (ATGenium) and overlapping oligonucleotides (NZYOligo designer), in order to allow automation of the high-throughput gene synthesis platform. The platform can assemble 96 synthetic genes encoding venom peptides simultaneously, with an error rate of 1.1 mutations per kb. To decrease the error rate associated with artificial gene synthesis, an error removal step using phage T7 endonuclease I was designed and integrated into the gene synthesis methodology. T7 endonuclease I was shown to be highly effective to specifically recognize and cleave DNA mismatches allowing a dramatically reduction of error frequency in large synthetic genes, from 3.45 to 0.43 errors per kb. Combining the knowledge acquired in the initial stages of the work, a comprehensive study was performed to investigate the influence of gene design, presence of fusion tags, cellular localization of expression, and usage of Tobacco Etch Virus (TEV) protease for tag removal, on the recombinant expression of disulfide-rich venom peptides in Escherichia coli. Codon usage dramatically affected the levels of recombinant expression in E. coli. In addition, a significant pressure in the usage of the two cysteine codons suggests that both need to be present at equivalent levels in genes designed de novo to ensure high levels of expression. This study also revealed that DsbC was the best fusion tag for recombinant expression of disulfide-rich peptides, in particular when expression of the fusion peptide was directed to the bacterial periplasm. TEV protease was highly effective for efficient tag removal and its recognition sites can tolerate all residues at its C-terminal, with exception of proline, confirming that no extra residues need to be incorporated at the N-terminus of recombinant venom peptides. This study revealed that E. coli is a convenient heterologous host for the expression of soluble and potentially functional venom peptides. Thus, this novel high-throughput gene synthesis platform was used to produce ~5,000 synthetic genes with a low error rate. This genetic library supported the production of the largest library of recombinant venom peptides constructed until now. The library contains 2736 animal venom peptides and it is presently being screened for the discovery of novel drug leads related to different diseases.