| Congresso Brasileiro de Microbiologia 2023 | Resumo: 684-1 | ||||
Resumo:Tissue Inhibitors of Metalloproteinases (TIMPS) is a family of endogenous proteins that inhibit the Matrix Metalloproteinases (MMPs), keeping the homeostasis of the body tissue. Among them, TIMP1 is a glycoprotein that presents two independently functional domains (N and C terminal), while the Ndomain inhibits the MMPs’ action, the C-domain can induce the formation of pro-MMPs. In mode to explore the therapeutic effect of TIMP1 in the treatment of metastatic cancer, the protein can be expressed only with its N-terminal domain. Hence, our work aims to express the recombinant protein N-TIMP1 (NT1) in Pichia pastoris and to optimize its expression/secretion using a bioreactor, firstly, the nucleotide and the amino acids sequences of the protein of interest (TIMP1) were obtained in the KEGG platform. These sequences were used to analysis the structure and based on its crystalline structure found in PDB the NT1 was designed. From this, computational analysis was performed to predict the interactions between NT1 and MMP2, analyze the structure of the protein and calculate the protein IP, through HDOCK, PyMol and ProtParam softwares, respectively. The resulting analysis of its interaction showed that NT1 interplay with MMP2 without the presence of the C-terminus domain presenting 3 extra amino acids in the interaction and a shortening of bonding compared to the interaction between the MMP2 and the whole TIMP1. This reduction in this distance shows a stronger substrate-enzyme interaction when the C-terminal domain is removed. The heterologous expression of NT1 gene was obtained from the synthetic and optimized sequence used as template to amplify the truncated fragment by PCR, which was inserted at the pPICZ expression vector by Gibson Assembly method. The recombinant cassete was transformed into Escherichia coli competent cells and the positive colonies, selected by Zeocin® had its plasmid purified and linearized in the restriction sites PmeI and BstX, these fragments were used to transform the P. pastoris yeast cells by electroporation. All the colonies, verified via colony PCR, were positive for NT1 gene and five colonies were randomly selected for the expression/secretion screening of NT1 protein analyzed by SDS-PAGE. The recombinant strain showing the capacity to produce high amount of total proteins (evaluated by Bradford assay), was selected for scale-up the production. The recombinant NT1 protein was purified by affinity
chromatography through 6x His-tag confirming the presence of NT1 in the
supernatant (Figure 1). The preliminary results showed that production of the
N-terminal portion of TIMP1 is relevant to obtain a protein that can function as a target therapy in the metastatic cascade in different types of cancer and as well as the utilization of P. pastoris as efficient system to produce NT1. The next steps will be the evaluation of NT1 activity using in vitro assays.
Palavras-chave: heterologous expression, metastasis treatment, pharmaceutical protein industry, TIMP Agência de fomento:Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) | |||||