Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast.
Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast.
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DOI：
10.1186/1472-6750-9-26
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发表时间：
2009-03-26
影响因子：
3.5
通讯作者：
Mayfield SP
中科院分区：
文献类型：
--
作者：
Muto M;Henry RE;Mayfield SP
在绿藻叶绿体中表达重组蛋白有望成为生产人类治疗性蛋白的平台。莱茵衣藻的叶绿体中表达了许多蛋白质，包括复杂的哺乳动物蛋白质，但其中许多蛋白质的积累水平明显低于内源叶绿体蛋白。我们研究了是否可以通过将重组报告蛋白荧光素酶基因融合到丰富的内源蛋白的羧基末端来促进重组蛋白的积累，内源蛋白是二磷酸核酮糖羧化酶(Rubisco LSU)的大亚基。此外，由于与内源性蛋白融合的重组蛋白几乎没有临床或商业价值，我们探索了将我们的重组蛋白改造为可在体内从内源性蛋白中切割的可能性。这一策略将不再需要进一步的体外加工步骤来生产所需的重组蛋白。为了实现这一点，在融合蛋白结构中的Rubisco LSU和荧光素酶编码区之间放置了首选氧化还蛋白(PreFd)的天然蛋白质加工位点。融合蛋白中的荧光素酶表达水平明显高于单独表达的荧光素酶。通过消除内源Rubisco大亚基基因(RbcL)，我们实现了在WT背景中荧光素酶积累相对于荧光素酶表达的进一步增加。重要的是，融合荧光素酶被蛋白水解性去除后，产生了接近野生型的功能性Rubisco全酶，而融合蛋白的荧光素酶活性几乎是单独表达的荧光素酶的33倍。这些数据证明了使用融合蛋白来促进重组蛋白在藻类叶绿体中积累的有效性，也表明工程蛋白水解点可以用来从内源融合伙伴中释放外源蛋白，从而提纯预期的成熟蛋白。这些结果表明，融合蛋白在藻类叶绿体中作为一种方法来增加难以表达的重组蛋白的积累是有效的。由于Rubisco在陆地植物和绿藻中普遍存在，这一策略也可以应用于更高层次的植物转基因表达系统。
Expression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins. A number of proteins have been expressed in the chloroplast of Chlamydomonas reinhardtii, including complex mammalian proteins, but many of these proteins accumulate to significantly lower levels than do endogenous chloroplast proteins. We examined if recombinant protein accumulation could be enhanced by genetically fusing the recombinant reporter protein, luciferase, to the carboxy-terminal end of an abundant endogenous protein, the large subunit of ribulose bisphosphate carboxylase (Rubisco LSU). Additionally, as recombinant proteins fused to endogenous proteins are of little clinical or commercial value, we explored the possibility of engineering our recombinant protein to be cleavable from the endogenous protein in vivo. This strategy would obviate the need for further in vitro processing steps in order to produce the desired recombinant protein. To achieve this, a native protein-processing site from preferredoxin (preFd) was placed between the Rubisco LSU and luciferase coding regions in the fusion protein construct. The luciferase from the fusion protein accumulated to significantly higher levels than luciferase expressed alone. By eliminating the endogenous Rubisco large subunit gene (rbcL), we achieved a further increase in luciferase accumulation with respect to luciferase expression in the WT background. Importantly, near-wild type levels of functional Rubisco holoenzyme were generated following the proteolytic removal of the fused luciferase, while luciferase activity for the fusion protein was almost ~33 times greater than luciferase expressed alone. These data demonstrate the utility of using fusion proteins to enhance recombinant protein accumulation in algal chloroplasts, and also show that engineered proteolytic processing sites can be used to liberate the exogenous protein from the endogenous fusion partner, allowing for the purification of the intended mature protein. These results demonstrate the utility of fusion proteins in algal chloroplast as a method to increase accumulation of recombinant proteins that are difficult to express. Since Rubisco is ubiquitous to land plants and green algae, this strategy may also be applied to higher plant transgenic expression systems.
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DOI：
10.1083/jcb.136.5.983
发表时间：
1997-03-10
期刊：
The Journal of cell biology
影响因子：
--
作者：
Akita M;Nielsen E;Keegstra K
通讯作者：
Keegstra K
影响因子：
7.2
作者：
Mayfield, SP;Schultz, J
通讯作者：
Schultz, J
影响因子：
2.8
作者：
Kasai, S;Yoshimura, S;Shinmyo, A
通讯作者：
Shinmyo, A
影响因子：
13.8
作者：
Manuell, Andrea L.;Beligni, Maria Veronica;Mayfield, Stephen P.
通讯作者：
Mayfield, Stephen P.
DOI：
10.1007/s004380051184
发表时间：
2000-04-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
Bateman, JM;Purton, S
通讯作者：
Purton, S