Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide.
Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide.
复制标题
DOI：
10.1371/journal.pone.0043349
复制
发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Mayfield SP
中科院分区：
文献类型：
--
作者：
Rasala BA;Lee PA;Shen Z;Briggs SP;Mendez M;Mayfield SP
微藻作为生产重组蛋白和新型代谢物的潜在低成本宿主，最近受到关注。然而，藻类作为工业平台的发展的主要障碍是来自核基因组的异源基因的表达差。在这里，我们描述了一种核表达策略，使用口蹄疫病毒2A自切割肽转录融合异源基因表达的抗生素耐药性莱茵衣藻。我们证明了用ble-2A-GFP转化的菌株具有博来霉素抗性，并且积累了高水平的GFP，所述GFP在FMDV 2A肽处被适当地“切割”，从而产生单体的胞质GFP，所述单体的胞质GFP容易通过凝胶内荧光分析或荧光显微镜检测。此外，我们使用我们的ble 2A核表达载体工程异源表达的工业酶，木聚糖酶。我们证明，连接xyn 1表达ble 2A表达相同的开放阅读框架导致一个显着的（约100倍）增加木聚糖酶活性的细胞裂解物相比，未连接的构建体。最后，通过在ble 2A和xyn 1编码区之间插入内源性分泌信号，我们能够靶向单体木聚糖酶进行分泌。本文所述的新型微藻核表达策略使得能够选择有效表达异源感兴趣基因的转基因株系，并且应该证明对于基础研究以及藻类生物技术是有价值的。
Microalgae have recently received attention as a potential low-cost host for the production of recombinant proteins and novel metabolites. However, a major obstacle to the development of algae as an industrial platform has been the poor expression of heterologous genes from the nuclear genome. Here we describe a nuclear expression strategy using the foot-and-mouth-disease-virus 2A self-cleavage peptide to transcriptionally fuse heterologous gene expression to antibiotic resistance in Chlamydomonas reinhardtii. We demonstrate that strains transformed with ble-2A-GFP are zeocin-resistant and accumulate high levels of GFP that is properly ‘cleaved’ at the FMDV 2A peptide resulting in monomeric, cytosolic GFP that is easily detectable by in-gel fluorescence analysis or fluorescent microscopy. Furthermore, we used our ble2A nuclear expression vector to engineer the heterologous expression of the industrial enzyme, xylanase. We demonstrate that linking xyn1 expression to ble2A expression on the same open reading frame led to a dramatic (∼100-fold) increase in xylanase activity in cells lysates compared to the unlinked construct. Finally, by inserting an endogenous secretion signal between the ble2A and xyn1 coding regions, we were able to target monomeric xylanase for secretion. The novel microalgae nuclear expression strategy described here enables the selection of transgenic lines that are efficiently expressing the heterologous gene-of-interest and should prove valuable for basic research as well as algal biotechnology.
登录
查看更多内容
影响因子：
5.5
作者：
Mayfield, SP;Franklin, SE
通讯作者：
Franklin, SE
影响因子：
11.6
作者：
Cerutti, H;Johnson, AM;Boynton, JE
通讯作者：
Boynton, JE
影响因子：
--
作者：
Lodha, Mukesh;Schulz-Raffelt, Miriam;Schroda, Michael
通讯作者：
Schroda, Michael
影响因子：
13.8
作者：
Manuell, Andrea L.;Beligni, Maria Veronica;Mayfield, Stephen P.
通讯作者：
Mayfield, Stephen P.
影响因子：
2.9
作者：
Ge, Yue;Antoulinakis, Evan G.;Johnson, Lain
通讯作者：
Johnson, Lain