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

Abstract

Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear whether this is because of few attempts or of limitations of the system that preclude expression of many proteins. Thus, we sought to assess the versatility of transgenic algae as a recombinant protein production platform. To do this, we tested whether the algal chloroplast could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, >50% expressed at levels sufficient for commercial production. Three expressed at 2%-3% of total soluble protein, while a forth protein accumulated to similar levels when translationally fused to a well-expressed serum amyloid protein. All of the algal chloroplast-expressed proteins are soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivity achieved demonstrate the utility of Chlamydomonas reinhardtii as a robust platform for human therapeutic protein production.

Figure 1. Introduction of the recombinant genes into the Chlamydomonas reinhardtii chloroplast genome

Schematic diagram of transformation vectors used, including relevant restriction sites. (A and B) pD1-Kan: Replacement of the endogenous psbA gene with the gene of interest (A), or with the gene of interest fused to the C-terminus of M-SAA (Manuell et al., 2007) (B). The kanamycin resistance gene aphA6 under the control of the atpA promoter and 5′ UTR is genetically linked to the gene of interest. Grey regions flanking the gene of interest and resistance gene corresponds to regions of the chloroplast genome used for homologous recombination between the insertion plasmid and the C. reinhardtii chloroplast genome. (C) Schematic diagram of p322 (Franklin et al., 2002) used to transform the genes of interest under the control of the atpA promoter and 5′ UTR and the rbcL 3′ UTR into the BamHI silent site near the psbA gene (Barnes et al., 2005). (A-C) All recombinant proteins were C-terminally fused to the 1 x FLAG-tag sequence (DYKDDDDKS) for western blotting and purification.

Figure 2. Identification of gene integration and isolation of homoplasmic strains

PCR using whole cell lysates as described in the Experimental Procedures. G: Gene specific PCR to show the presence of the corresponding recombinant gene in the transformants. H: PCR to show homoplasmicity of the clones. Each reaction contains two sets of primers (▶ ◀), one that amplifies an internal control gene (16S rRNA) to demonstrate that the PCR reactions worked, and can be seen in all lanes (H-C), and the other primer set amplifies the region of the genome that was targeted for integration and thus the parent strain shows a band whereas homoplasmic transformants do not (H-I).

Figure 3. Accumulation of recombinant proteins in transgenic lines

Strains were grown under the same conditions and harvested for western blot as described in the Experimental Procedures. Equal amounts of total protein were loaded in each lane (20 μg for A and C, 40 μg for B). Western blots were probed with anti-FLAG antibody conjugated to Horse Radish Peroxidase. (A) Protein accumulation in strains when the corresponding genes were expressed from the psbA promoter and UTRs. (B) Protein accumulation when the corresponding genes were expressed from the atpA promoter and UTRs. (C) Protein accumulation of the SAA fusion proteins from the psbA promoter. Note, short exposure times were routinely used for (A) and (C), while much longer exposure times (several hours) were required to visualize the bands in (B).

Figure 4. Quantitation of protein accumulation

Percent total soluble protein was determined for 14FN3, VEGF, and HMGB1 by loading 10 or 20 μg of soluble lysate from expression strains onto a SDS-PAGE gel alongside a serial dilution of highly pure HMGB1. Western blots were performed using anti-FLAG-HRP antibody.

Figure 5. Analysis of mRNA levels for psbA and atpA constructs

mRNA levels of the seven recombinant genes under the control of the psbA and atpA promoters. Fold change determined using the Pfaffl method (Pfaffl, 2001) to take into account differing PCR efficiencies with the different gene-specific primer pairs used. Note, atpA-EPO yielded the lowest level of mRNA, so all mRNA levels were calculated as fold change relative to atpA-EPO. The recombinant protein expressing lines are indicated (*).

Figure 6. Affinity purification of algal-expressed therapeutic proteins

Coomassie staining (top left panel) and western blotting (boxed bottom panel) of (A) 14FN3, (B) VEGF, and (C) HMGB1 purifications are shown. Lanes from left to right are the following fractions: insoluble fraction (Ins), total soluble protein (TSP), column flow through (Flow), and the eluate (Elu). Equal volumes of Ins, TSP and Flow are loaded per lane. 3 μg of purified protein (Elu) is loaded on each coomassie gel, while 500 ng of Elu is loaded on each western blot. Right panels correspond to the MADLI-TOF MS results for each purified protein. Asterisk (*) in B indicates predicted dimer form of VEGF.

Figure 7. Bioactivity of VEGF

(A) Concentration of intact VEGF in the purified protein was assessed by comparison with bacteria-derived VEGF in a sandwich ELISA. (B) Competitive binding to the VEGF receptor was assayed by detecting binding of a fixed concentration of algal VEGF to VEGFR-coated wells in the presence of varying concentrations of bacteria-derived VEGF.

Figure 8. Bioactivity of HMGB1

Graph of the results from the fibroblast chemotaxis assay, as measured by the number of mouse (A) or pig (B) fibroblasts migrating towards the indicated chemokine is shown. Bioactivity of algal-expressed HMGB1 (Scripps) compared to commercial HMGB1 (Bio3), and to the controls mouse VEGF (A) or pig PDGF (B). Data represents mean and standard deviations of each treatment condition.