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

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.

Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii

“…, 2004; Butt et al. , 2005) and plant and algal chloroplasts (Streatfield, 2007; Muto et al. , 2009).…”

“…These include reporter proteins (Franklin et al. , 2002; Mayfield and Schultz, 2004; Muto et al. , 2009), a large complex mammalian single‐chain antibody (Mayfield et al.…”

Liquid culture of microalgae in a photobioreactor (PBR) based on oscillatory baffled reactor (OBR) technology – A feasibility study

“…Numerous studies have reported the successful use of the microalga Chlamydomonas reinhardtii for production of therapeutic proteins , Muto et al, 2009, Rosales-Mendoza et al, 2012, Tran et al, 2009, Mayfield, 2013. Of particular interest are synthetic fusion proteins that combine the characteristics from different molecules to provide an enhanced physiological response (Mayfield, 2013).…”

Random mutagenesis and precise gene editing technologies: applications in algal crop improvement and functional genomics

“…Alternatively, transgenic algae can be used as bioreactors for the production of pharmaceuticals (Hawkins & Nakamura, 1999;Geng et al, 2003;Mayfield et al, 2003;Sun et al, 2003). For example, C. reinhardtii chloroplasts have been established as factories for the production of recombinant proteins Mayfield et al, 2007;Muto et al, 2009;Rasala et al, 2010) and offer efficient systems for high yield production of complex proteins which were properly folded into functional molecules such as recombinant antibodies Tran et al, 2009) and vaccines (Surzycki et al, 2009). Moreover, the development of efficient target genome editing methods to specifically knock down or knock out genes could be exploited for functional genomic analyses to enhance understanding of algal biology.…”