Malla Ashwini, Rosales-Mendoza Sergio, Phoolcharoen Waranyoo, Vimolmangkang Sornkanok
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
Research Unit for Plant-Produced Pharmaceuticals, Chulalongkorn University, Bangkok, Thailand.
Front Plant Sci. 2021 Apr 7;12:650820. doi: 10.3389/fpls.2021.650820. eCollection 2021.
The increase in the world population, the advent of new infections and health issues, and the scarcity of natural biological products have spotlighted the importance of recombinant protein technology and its large-scale production in a cost-effective manner. Microalgae have become a significant promising platform with the potential to meet the increasing demand for recombinant proteins and other biologicals. Microalgae are safe organisms that can grow rapidly and are easily cultivated with basic nutrient requirements. Although continuous efforts have led to considerable progress in the algae genetic engineering field, there are still many hurdles to overcome before these microorganisms emerge as a mature expression system. Hence, there is a need to develop efficient expression approaches to exploit microalgae for the production of recombinant proteins at convenient yields. This study aimed to test the ability of the DNA geminiviral vector with Rep-mediated replication to transiently express recombinant proteins in the freshwater microalgal species and using mediated transformation. The SARS-CoV-2 receptor binding domain (RBD) and basic fibroblast growth factor (bFGF) are representative antigen proteins and growth factor proteins, respectively, that were subcloned in a geminiviral vector and were used for nuclear transformation to transiently express these proteins in and . The results showed that the geminiviral vector allowed the expression of both recombinant proteins in both algal species, with yields at 48 h posttransformation of up to 1.14 μg/g RBD and 1.61 ng/g FGF in and 1.61 μg/g RBD and 1.025 ng/g FGF in . Thus, this study provides a proof of concept for the use of DNA viral vectors for the simple, rapid, and efficient production of recombinant proteins that repress the difficulties faced in the genetic transformation of these unicellular green microalgae. This concept opens an avenue to explore and optimize green microalgae as an ideal economically valuable platform for the production of therapeutic and industrially relevant recombinant proteins in shorter time periods with significant yields.
世界人口的增长、新感染和健康问题的出现以及天然生物制品的稀缺，凸显了重组蛋白技术及其以经济高效方式进行大规模生产的重要性。微藻已成为一个极具潜力的重要平台，有望满足对重组蛋白和其他生物制品日益增长的需求。微藻是安全的生物体，生长迅速，只需基本营养需求即可轻松培养。尽管持续的努力已在藻类基因工程领域取得了相当大的进展，但在这些微生物成为成熟的表达系统之前，仍有许多障碍需要克服。因此，需要开发高效的表达方法，以便利用微藻以适宜的产量生产重组蛋白。本研究旨在测试具有Rep介导复制功能的DNA双生病毒载体在淡水微藻物种中通过介导转化瞬时表达重组蛋白的能力。严重急性呼吸综合征冠状病毒2（SARS-CoV-2）受体结合域（RBD）和碱性成纤维细胞生长因子（bFGF）分别是代表性的抗原蛋白和生长因子蛋白，它们被亚克隆到双生病毒载体中，并用于核转化，以便在两种微藻中瞬时表达这些蛋白。结果表明，双生病毒载体使两种重组蛋白在两种藻类物种中均得以表达，转化后48小时，在一种微藻中的产量高达1.14μg/g RBD和1.61ng/g FGF，在另一种微藻中的产量为1.61μg/g RBD和1.025ng/g FGF。因此，本研究为使用DNA病毒载体简单、快速且高效地生产重组蛋白提供了概念验证，克服了这些单细胞绿色微藻基因转化中面临的困难。这一概念为探索和优化绿色微藻作为理想的经济有价值平台开辟了一条途径，以便在更短的时间内以显著的产量生产治疗性和工业相关的重组蛋白。
