Site-specific incorporation of non-natural amino acids in proteins: development of new incorporation technologies and the use in the study of proteins

• Grant number: 14/20847-0
• Support Opportunities: Regular Research Grants
• Start date: September 01, 2015
• End date: August 31, 2017
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Vitor Marcelo Silveira Bueno Brandão de Oliveira
• Grantee: Vitor Marcelo Silveira Bueno Brandão de Oliveira
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated researchers: Jaap Broos ; Luciano Puzer

Abstract

In the last 4 years we have focused our research in site-specific incorporation techniques of non-natural amino acids in proteins. For this purpose two main methodologies have been developed: -the first one is the introduction of tryptophan analogs (5-fluor-Trp, 5-hidroxi-Trp, 7-aza-Trp, 5-bromo-Trp and 5-nitro-Trp) in the place of the Trp residues, by using E. coli strains compatible with expression vectors that need the T7 polimerase (expression of the target protein due to the strong T7 promoter). The best known and most used vectors of this type are the commercially available pET vectors (Merck Millipore - former Novagen); -the second one is the introduction of non-natural amino acids thorough an orthogonal tRNA - tRNA-sinthetase pair, modified to allow the incorporation of the p-nitro-phenylalanine amino acid (fluorescence acceptor probe) in the place coded by the amber stop codon (UAG), introduced in the target protein gene at a pre-designed site by directed mutagenesis; Initially, these technologies will be applied in structure-activity studies of the thimet oligopeptidase aiming the detection of conformational changes, predicted to occur upon ligand binding at the active site of this enzyme. However, the choice of this peptidase as the initial target is due to the non-natural amino acids that are spectroscopic probes, but also mainly to our previous experience in working with this peptidase, including the production of many mutants. Besides, as we have successfully incorporated Trp analogs as well as the p-nitro-Phe in the recombinant thimet oligopeptidase, we are planning to expand this technology: to the study of other proteins and to apply in other biotechnological purposes. Examples: the study of other peptidases that also undergo large conformational changes during catalysis; to label enzyme protein inhibitors such as serpins or to label recombinant antibodies that can be used in detection/localization assays even in vivo or also in new clinical diagnosis methods. Furthermore, new plasmid constructions are being carried out aiming the bio-incorporation of other non-natural amino acids: one amino acid with a coumarin side chain (fluorescence probe); the p-azide-phenylalanine and the p-benzoyl-phenylalanine that are photo-activable reactive groups that can be used for specific protein labeling or in protein-protein interaction studies. (AU)

Scientific publications

(5)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

BELIZARIO, JOSE E.. Cancer Risks Linked to the Bad Luck Hypothesis and Epigenomic Mutational Signatures. EPIGENOMES, v. 2, n. 3, SEP 2018. (12/02497-7, 14/20847-0)

OLIVEIRA-SOUZA, WELLINGTON P.; BRONZE, FELLIPE; BROOS, JAAP; MARCONDES, MARCELO F. M.; OLIVEIRA, VITOR. On the efficient bio-incorporation of 5-hydroxy-tryptophan in recombinant proteins expressed in Escherichia coli with T7 RNA polymerase-based vectors. Biochemical and Biophysical Research Communications, v. 492, n. 3, p. 343-348, OCT 21 2017. (14/20847-0, 11/20941-9, 14/00661-0)

GILIO, JOYCE M.; MARCONDES, MARCELO F.; FERRARI, DEBORA; JULIANO, MARIA A.; JULIANO, LUIZ; OLIVEIRA, VITOR; MACHADO, MAURICIO F. M.. Processing of metacaspase 2 from Trypanosoma brucei (TbMCA2) broadens its substrate specificity. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, v. 1865, n. 4, p. 388-394, APR 2017. (15/11190-0, 14/20847-0, 12/50191-4)

ICIMOTO, MARCELO Y.; FERREIRA, JULIANA C.; YOKOMIZO, CESAR H.; BIM, LARISSA V.; MAREM, ALYNE; GILIO, JOYCE M.; OLIVEIRA, VITOR; NANTES, ISELI L.. Redox modulation of thimet oligopeptidase activity by hydrogen peroxide. FEBS OPEN BIO, v. 7, n. 7, p. 1037-1050, JUL 2017. (12/07456-7, 14/20847-0, 11/05986-6)

BELIZARIO, JOSE E.; SIRCILI, MARCELO P.. Novel biotechnological approaches for monitoring and immunization against resistant to antibiotics Escherichia coli and other pathogenic bacteria. BMC Veterinary Research, v. 16, n. 1, NOV 2 2020. (12/02497-7, 14/20847-0, 10/06707-0, 08/05519-6)