Humanization of the glycosylation system of Pichia Pastoris via CRISPR-Cas9 technique aiming the expression of therapeutical recombinant proteins

• Grant number: 15/25055-8
• Support Opportunities: Regular Research Grants
• Duration: May 01, 2016 - October 31, 2018
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Paulo Lee Ho
• Grantee: Paulo Lee Ho
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

The production of recombinant therapeutical proteins represents one of the most productive industries in the area of molecular medicine nowadays, comprising complex molecules of high value. These proteins normally present a glycosylation pattern proper of humans, having great importance in protein folding, stability and biological activity. Seeking to reproduce the human glycosylation pattern in yeast, this project aims to establish a Pichia pastoris strain with a specific human glycosylation pattern through the deletion of two genes involved in yeast glycosylation system, alg3 and och1, responsible for the posterior hiper-mannosylation characteristic of this organism. This way, it is possible to achieve a common intermediate to the human glycosylation pathway. The genome editing will be performed using the CRISPR-Cas9 technique, a method that has revolutionized the genetic engineering field in the past two years. This system, which belongs to prokaryotes' adaptive immune system, was adapted to genome editing in higher organisms and allows, in a simple manner, gene insertion and deletion using a single enzyme, Cas9, which performs double strand cleavage of the DNA guided by a RNA duplex. In order to test the efficiency of this new humanized glycosylation system, the expression of the recombinant protein glucocerebrosidase (GCR) was suggested as a model due to its importance and economical relevance. This enzyme deficiency results in different types of Gaucher disease, some of them can be treated with enzymatic reposition therapy. The active form of recombinant GCR employed in therapy presents exposed mannose residues in its glycosylation pattern, and it is this profile that is intended to be reproduced in P. pastoris strains with a humanized glycosylation system. The success in the establishment of this yeast expression system for recombinant protein production will allow the development of a national platform to the production of other enzymes to enzymatic replacement therapy and other proteins of biotechnological interest, representing an initial project to the implementation of genomic editing systems and synthetic biology at Butantan Institute. (AU)

Scientific publications

(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)

AKAMATSU, MILENA APETITO; SAKIHARA, VITOR ANSELMO; CARVALHO, BIANCA PEREIRA; ABRANTES, ALINE DE PAIVA; TAKANO, MARIA A. SAKAUCHI; ADAMI, EDUARDO ALFREDO; YONEHARA, FERNANDO SEIJI; CARNEIRO, PATRICIA DOS SANTOS; RICO, STEFANNI; SCHANOSKI, ALESSANDRA; et al. Preparedness against pandemic influenza: Production of an oil-in-water emulsion adjuvant in Brazil. PLoS One, v. 15, n. 6, JUN 3 2020. (15/25055-8)

SOARES-SCHANOSKI, ALESSANDRA; BAPTISTA CRUZ, NATALIA; DE CASTRO-JORGE, LUIZA ANTUNES; HOMEM DE CARVALHO, RENAN VILLANOVA; DOS SANTOS, CLIOMAR ALVES; DA ROS, NANCY; OLIVEIRA, URSULA; COSTA, DANUZA DUARTE; SIMOES DOS SANTOS, CECILIA LUIZA; CUNHA, MARIELTON DOS PASSOS; et al. Systems analysis of subjects acutely infected with the Chikungunya virus. PLOS PATHOGENS, v. 15, n. 6, JUN 2019. (12/19278-6, 13/07467-1, 15/25055-8, 13/08216-2, 16/08204-2, 17/50137-3)