Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast

Machiko Muto,Ryan E Henry,Stephen P Mayfield

doi:10.1186/1472-6750-9-26

Machiko Muto, Ryan E Henry + Show 1 more

Open Access

https://doi.org/10.1186/1472-6750-9-26

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Abstract

BackgroundExpression 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.ResultsThe 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.ConclusionThese 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.

Highlights

Expression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins
We demonstrate that Rubisco LSU protein derived solely from the fusion protein construct is completely functional, as evidenced by normal photosynthesis in transformants where the endogenous Rubisco large subunit gene (rbcL) gene has been eliminated
We had previously constructed the vector as an internal standard for replacing the endogenous psbA gene, thereby generating a non-photosynthetic strain, and showed that expression of the luxCt reporter using this rbcL promoter and 5' untranslated region (UTR) achieved recombinant protein accumulation to about 0.05% of total protein [7]

Summary

Introduction

Expression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins. 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. The use of plants and green algal chloroplasts for transgenic protein expression has some practical advantages compared to that of nuclear expression. These advantages include the absence of gene silencing, the ability to precisely target the gene of interest to specific regions of the chloroplast genome by homologous recombination, the potential for robust expression of heterologous proteins, rapid generation of stable transgenic lines, simple promoter and expression elements, and limited post-translational modifications to the recombinant protein [2,3,4]. Methods for transforming the Chlamydomonas chloroplast genome are relatively simple [8,9,10], and chloroplast transformants can be selected through co-transformation with DNA conferring resistance to antibiotics [11,12,13] or through phototrophic rescue [10]

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