Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae

Abstract

Eukaryotic green algae have become an increasingly popular platform for recombinant proteins production. In particular, Chlamydomonas reinhardtii, has garnered increased attention for having the necessary biochemical machinery to produce vaccines, human antibodies and next generation cancer targeting immunotoxins. While it has been shown that chloroplasts contain chaperones, peptidyl prolylisomerases and protein disulfide isomerases that facilitate these complex proteins folding and assembly, little has been done to determine which processes serve as rate-limiting steps for protein accumulation. In other expression systems, as Escherichia coli, Chinese hamster ovary cells, and insect cells, recombinant protein accumulation can be hampered by cell's inability to fold the target polypeptide into the native state, resulting in aggregation and degradation. To determine if chloroplasts' ability to oxidize proteins that require disulfide bonds into a stable conformation is a rate-limiting step of protein accumulation, three recombinant strains, each expressing a different recombinant protein, were analyzed. These recombinant proteins included fluorescent GFP, a reporter containing no disulfide bonds; Gaussia princeps luciferase, a luminescent reporter containing disulfide bonds; and an immunotoxin, an antibody-fusion protein containing disulfide bonds. Each strain was analyzed for its ability to accumulate proteins when supplemented with selenocystamine, a small molecule capable of catalyzing the formation of disulfide bonds. Selenocystamine supplementation led to an increase in luciferase and immunotoxin but not GFP accumulation. These results demonstrated that selenocystamine can increase the accumulation of proteins containing disulfide bonds and suggests that a rate-limiting step in chloroplast protein accumulation is the disulfide bonds formation in recombinant proteins native structure.

Figure 1

DNA gene constructs, site of integration and identification of transformants in Chlamydomonas reinhardtii chloroplast. a Transformation vector containing the genes of interest: Gluc (Luciferase from Gaussia princeps), or GFP (Green Fluorescent Protein from Aequorea vcitoria), or Immunotoxin (αCD22CH23PE40 (Tran et al. 2013b)). Regions of the chloroplast genome were positioned at both ends of the transformation vector to allow homologous integration of the whole transformation cassette into the chloroplast genome (w1.1). b, c Colony PCR amplification to confirm the presence of recombinant genes of interest, Gluc—Luciferase from Gaussia princeps (b) or GFP—Green Fluorescent Protein (c). b, c Lanes 1, 2 and 3 show three different gene positive colonies (dotted arrow). d, e Colony PCR amplification to check homoplasmicity by using primers from the genome to verify the absence of the second band (dashed arrow), which indicates the replacement of SAA by our recombinant genes. The black arrow shows the amplified control PCR product. d, e Lanes 1 and 3 show homoplasmic colonies, and lane 2 a control. All the arrows (dotted, dashed and black) are also marked (a) to indicate the gene amplified regions.

Figure 2

Growth curves of recombinant Chlamydomonas reinhardtii strains cultivations treated with increasing concentrations of selenocystamine. a Cell concentration (cells mL⁻¹) as a function of time (h), for recombinant C. reinhardtii—Gluc (strain expressing Gaussia luciferase) cultivations containing: 0.0, 0.1, 1.0, 2.0, 5.0, 10.0, 25.0 and 100.0 µM selenocystamine final concentration in the culture medium, compared to control w1.1 (psbA knockout). b Cell concentration (cells mL⁻¹) as a function of time (h), for recombinant C. reinhardtii—GFP (strain expressing green fluorescent protein) cultivations containing: 0.0, 0.1, 1.0, 2.0, 5.0, 10.0, 25.0 and 100.0 µM selenocystamine final concentration in the culture medium, compared to control w1.1 (psbA knockout). Error bars were calculated from triplicate average values of three different experiments.

Figure 3

Luminescence of recombinant C. reinhardtii—Gluc (strain expressing Gaussia luciferase protein) cultivations supplemented with selenocystamine. This oxidative molecule was supplemented to obtain the following final concentrations: 0.0, 0.1, 1.0, 2.0, 5.0, 10.0, 25.0 and 100.0 µM, and these were compared to control w1.1. a 0 h of cultivation; b 8 h; c 24 h; d 48 h; e 72 h; f 96 h. All error bars were calculated by using the triplicate average values of different experiments, and the values were equalized to total protein concentration.

Figure 4

Fluorescence of recombinant C. reinhardtii—GFP (strain expressing green fluorescent protein) cultivations supplemented with selenocystamine. This oxidative molecule was supplemented to obtain the following final concentrations: 0.0, 0.1, 1.0, 2.0, 5.0, 10.0, 25.0 and 100.0 µM, and these were compared to control w1.1. a 0 h of cultivation; b 8 h; c 24 h; d 48 h; e 72 h; f 96 h. RFU relative fluorescence unit. All error bars were calculated by using the triplicate average values, which were also equalized to cell concentration measurements.

Figure 5

Immunotoxin (αCD22CH23PE40) expression in Chlamydomonas reinhardtii chloroplast. a The bar graph shows the percentage of expression for samples withdrawn after 48 h of cultivation in each experiment, which had 2.0 µM final concentration of cystamine (Cys) or selenocystamine (Secyst) compared to control (w1.1). All error bars were calculated by using the triplicate average values, which were also equalized to total protein concentration. b Calibration curve used for quantitative ELISA assay. Absorbance is shown as a function of recombinant protein αCD22CH23PE40 expression in percentages (0.0, 0.5, 1.0 and 5.0%).