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建立绿藻作为重组蛋白生产的平台。

Establishing the green algae as a platform for recombinant protein production.

作者信息

Kang Kalisa, do Espirito Santo Évellin, Diaz Crisandra Jade, Oliver Aaron, Saxton Lisa, May Lauren, Mayfield Stephen, Molino João Vitor Dutra

机构信息

Department of Molecular Biology, School of Biological Sciences, University of California San Diego, La Jolla, CA, United States of America.

Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, Sao Pãulo, Brazil.

出版信息

bioRxiv. 2024 Oct 25:2024.10.25.618925. doi: 10.1101/2024.10.25.618925.

Abstract

, a genetically close relative of the model green alga , shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between and , this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta's capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with , exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to . For xylanase expression and secretion, both and showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, showed comparable PHL7 activity levels to , as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between and as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize 's potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.

摘要

作为模式绿藻在基因上的近亲,[具体物种名未给出]在重组蛋白表达宿主方面显示出巨大潜力。由于[具体物种名未给出]与[另一物种名未给出]之间存在密切的遗传关系,该物种为增进我们对光合生物的理解提供了额外的参考点,也为生物技术应用提供了一个潜在的新候选者。本研究调查了[具体物种名未给出]表达三种重组蛋白的能力:荧光蛋白mCherry、半纤维素降解酶木聚糖酶和塑料降解酶PHL7。我们还研究了将蛋白表达靶向该藻类各种细胞区室的能力,包括细胞质、分泌途径、细胞质膜和细胞壁。与[另一物种名未给出]直接比较时,[具体物种名未给出]在重组蛋白生产方面表现出独特但显著的能力。用编码mCherry的载体进行细胞转化显示,与[另一物种名未给出]相比,[具体物种名未给出]产生的荧光水平高出约3.5倍,免疫印迹强度增加了3.7倍。对于木聚糖酶的表达和分泌,[具体物种名未给出]和[另一物种名未给出]都显示出相似的分泌能力和酶活性,木聚糖降解率相当,突出了木聚糖酶在微藻中表达的工业适用性。最后,通过聚酯聚氨酯悬浮液Impranil DLN的体外降解证明,[具体物种名未给出]显示出与[另一物种名未给出]相当的PHL7活性水平。最后,我们还探索了细胞融合在[具体物种名未给出]和[另一物种名未给出]之间产生遗传杂种的潜力,以此作为增强表型多样性和增加遗传变异的一种手段。我们能够产生遗传融合体,其可以将重组蛋白基因以及相关的选择标记基因交换到重组后代中。这些发现强调了[具体物种名未给出]作为重组蛋白生产的强大平台以及作为更好地理解微藻生物学的有力工具的潜力。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/964b/11527144/99d2ae4f0e6f/nihpp-2024.10.25.618925v1-f0001.jpg

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