Improving recombinant protein production in the Chlamydomonas reinhardtii chloroplast using vivid Verde Fluorescent Protein as a reporter.
Improving recombinant protein production in the Chlamydomonas reinhardtii chloroplast using vivid Verde Fluorescent Protein as a reporter.
复制标题
DOI：
10.1002/biot.201400566
复制
发表时间：
2015-08
影响因子：
4.7
通讯作者：
Purton S
中科院分区：
文献类型：
--
作者：
Braun-Galleani S;Baganz F;Purton S
微藻具有易于培养、无病原菌、下游加工成本低等优点，具有合成高价值重组蛋白的潜力。然而，与其他微生物平台相比，目前的重组蛋白水平较低，只有少数几种微藻可以稳定插入转基因。我们探索了不同的策略，旨在提高莱茵衣藻叶绿体的生长速度和重组蛋白产量。在天然ATPA启动子/5‘非编码区元件的调控下，表达了一种新的荧光蛋白VFP。Western blotting检测到VFP水平，当与编码大肠杆菌Spy伴侣的基因共表达时，观察到蛋白水平增加。我们利用这些转化系研究了温度、光照和培养基对重组蛋白生产和细胞生长的影响。通过流式细胞仪对VFP水平和荧光进行评估，可以确定在混合营养模式下30°C的改进培养条件。在这些条件下对具有潜在商业价值的抗菌素内溶酶(CPL-1)的积累进行了测试，观察到VFP获得的结果不容易被CPL-1复制。本研究表明重组蛋白的表达是产物特异性的，需要单独优化。
Microalgae have potential as platforms for the synthesis of high‐value recombinant proteins due to their many beneficial attributes including ease of cultivation, lack of pathogenic agents, and low‐cost downstream processing. However, current recombinant protein levels are low compared to other microbial platforms and stable insertion of transgenes is available in only a few microalgal species. We have explored different strategies aimed at increasing growth rate and recombinant protein production in the Chlamydomonas reinhardtii chloroplast. A novel fluorescent protein (vivid Verde Fluorescent Protein, VFP) was expressed under the control of the native atpA promoter/5'UTR element. VFP levels were detected by western blotting, with increased protein levels observed when co‐expressed with a gene encoding the Escherichia coli Spy chaperone. We used these transformant lines to study the effect of temperature, light and media on recombinant protein production and cell growth. VFP levels and fluorescence, assessed by flow cytometry, allowed a determination of improved cultivation conditions as 30°C under mixotrophic mode. These conditions were tested for the accumulation of an antimicrobial endolysin (Cpl‐1) of potential commercial interest, observing that the outcome obtained for VFP could not be easily replicated for Cpl‐1. This study suggests that recombinant protein expression is product‐specific and needs to be optimized individually.