A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65)

Abstract

Background: Human glutamic acid decarboxylase 65 (hGAD65) is a key autoantigen in type 1 diabetes, having much potential as an important marker for the prediction and diagnosis of type 1 diabetes, and for the development of novel antigen-specific therapies for the treatment of type 1 diabetes. However, recombinant production of hGAD65 using conventional bacterial or mammalian cell culture-based expression systems or nuclear transformed plants is limited by low yield and low efficiency. Chloroplast transformation of the unicellular eukaryotic alga Chlamydomonas reinhardtii may offer a potential solution.

Results: A DNA cassette encoding full-length hGAD65, under the control of the C. reinhardtii chloroplast rbcL promoter and 5'- and 3'-UTRs, was constructed and introduced into the chloroplast genome of C. reinhardtii by particle bombardment. Integration of hGAD65 DNA into the algal chloroplast genome was confirmed by PCR. Transcriptional expression of hGAD65 was demonstrated by RT-PCR. Immunoblotting verified the expression and accumulation of the recombinant protein. The antigenicity of algal-derived hGAD65 was demonstrated with its immunoreactivity to diabetic sera by ELISA and by its ability to induce proliferation of spleen cells from NOD mice. Recombinant hGAD65 accumulated in transgenic algae, accounts for approximately 0.25-0.3% of its total soluble protein.

Conclusion: Our results demonstrate the potential value of C. reinhardtii chloroplasts as a novel platform for rapid mass production of immunologically active hGAD65. This demonstration opens the future possibility for using algal chloroplasts as novel bioreactors for the production of many other biologically active mammalian therapeutic proteins.

Figure 1

Schematic diagram of the hGAD65 chloroplast expression vector pXW-GAD-His and the site of integration of the transgene cassette into the chloroplast genome. The hGAD65 expression cassette consists of the C. reinhardtii chloroplast rbcL promoter and 5'UTR upstream of the transgene followed by the rbcL 3'UTR. The transgene cassette was inserted into plasmid vector p322 that contains a cloned 5.7 kb (EcoRI/XhoI fragment) inverted repeat of C. reinhardtii chloroplast DNA, resulting in pXW-GAD-His. The restriction sites used for cloning are indicated. Primers GAD-1/GAD-2 corresponding to the 5' and 3' ends of hGAD65 were used for PCR analysis to confirm the presence of the transgene in transformants, with the expected product size indicated. Primers CP3/CP4 complementary to sequences lying just outside the inverted repeat region of the C. reinhardtii chloroplast DNA were used to determine the site-specific integration of the transgene cassette into the C. reinhardtii chloroplast genome by PCR. The site-specific integration of the transgene cassette was additionally determined by PCR using GAD-1/CP3 and CP4/GAD-2 primer pairs. The regions for homologous recombination are indicated by the crosses. Selection of C. reinhardtii transformants was based on resistance to spectinomycin that was provided by co-transformation with plasmid p228 that contains the 16S rRNA gene conferring spectinomycin resistance [38].

Figure 2

PCR analysis of C. reinhardtii transformants. (A) hGAD65 specific primers GAD-1 and GAD-2 generated PCR products using total algal DNA as template. Lanes 1 to 5, independent C. reinhardtii transformants; PC, positive control (hGAD65 containing plasmid pXW-GAD-His). C. reinhardtii wild-type strain 137c DNA was used as a negative control. PCR products are indicated by the arrow. (B) Chloroplast specific primers CP3 and CP4 generated PCR products using total algal DNA as a template. Lanes 1 to 2, representative C. reinhardtii transformants; C. reinhardtii wild-type strain 137c DNA was used as a negative control. PCR products are indicated by the arrows. (C) The CP3/GAD-1 or CP4/GAD-2 primer set generated PCR products. Lane 1, representative C. reinhardtii transformant. C. reinhardtii wild-type strain 137c DNA was used as a negative control. PCR products are indicated by the arrows.

Figure 3

RT-PCR detection of hGAD65 mRNA expression in C. reinhardtii transformants. Total RNA was isolated from C. reinhardtii wild-type strain 137c and transformants. RNA was transcribed to cDNA as described in Methods. Resulting cDNA was amplified using primers specific to hGAD65. Lanes 1 to 4, PCR amplification of cDNA from independent transformants. PCR amplification of cDNA from wild-type strain 137c was used as a negative control. PC, positive control (direct PCR amplification of DNA from plasmid pXW-GAD-His). PCR products are indicated by the arrow.

Figure 4

Western blot detection of recombinant hGAD65 from total soluble protein extracted from chloroplast transgenic algal lines. Samples containing ~50 ng of commercial hGAD65 or 5 μg of total algal protein were separated by 15% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a polyvinylidene difluoride membrane and probed with rabbit anti-GAD65 primary antibody followed by horseradish peroxidase conjugated goat anti-rabbit secondary antibody. Standard, insect cell-derived hGAD65. Lanes 1 to 2, protein extracts from two representative algal transformants. Protein extracts from wild-type strain 137c were used as a negative control. Arrows indicate the position of algal-derived hGAD65.

Figure 5

Expression levels as percentages of hGAD65 in total soluble protein (TSP) of hGAD65 expressing C. reinhardtii transformants. The expression level was determined by hGAD65 specific ELISA as described in detail in methods. hGAD-1 and hGAD-2, two independent algal transformants; WT, untransformed wild type C. reinhardtii 137c strain.

Figure 6

Demonstration of immunoreactivity with NOD mouse diabetic sera to algal-derived hGAD65. Purified algal-derived hGAD65 was coated onto 96-well plates and incubated with serum samples from diabetic NOD mice (n = 3) and reference BALB/c mice (n = 3). Bound anti-GAD antibody was detected with HRP-conjugated anti-mouse IgG. Error bars represent the standard deviation. ** above bars indicates a significant difference from reference BALB/c sera (p < 0.05).

Figure 7

Spleen cell proliferation responses to algal-derived hGAD65. Individual 8-week-old NOD mice were tested for spontaneous proliferation of splenocytes to insect cell-derived hGAD65 standard, algal-derived hGAD65 or to the unrelated protein antigen ovalbumin (OVA). Error bars represent the standard deviation. ** above bars indicates a significant difference from OVA and medium alone (p < 0.05).