Expression and assembly of a fully active antibody in algae

Abstract

Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two C. reinhardtii chloroplast promoters and 5' and 3' RNA elements. This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes in vivo. Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification. We further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5' and 3' elements used to drive expression of the antibody gene. These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain.

Figure 1

Synthesis of a lsc antibody gene optimized for C. reinhardtii chloroplast expression. (A) The amino acid sequence is indicated by single-letter code and the heavy chain region is boxed and shaded with specific domains indicated by open arrows. The light chain variable region is boxed but unshaded. The complementarity-determining regions (CDR) are indicated by heavy boxes, and the Flag tag is indicated under the amino acid sequence. The linker region is unboxed. (B) Schematic diagram of the lsc protein as a dimer; the heavy chain is gray, the CDRs are black, and the linker and hinge region are indicated by a thin line. Cysteine residues capable of forming disulfide bonds are indicated as a line between the heavy chain constant regions, and the flag epitope is indicated.

Figure 2

Restriction maps of HSV8-lsc genes for expression in C. reinhardtii chloroplasts. (A) Relevant restriction sites delineating the rbcL 5′ UTR (BamHI/NdeI), the HSV8 coding region and flag tag (NdeI/XbaI), and the rbcL 3′ UTR (XbaI/BamHI), as well as relevant restriction sites of the atpA 5′ UTR (BamHI/NdeI), the HSV8 coding region and flag tag (NdeI/XbaI), and the rbcL 3′ UTR (XbaI/BamHI) are shown. (B) Restriction map showing the site of integration of the HSV8-lsc genes into plasmid p322 for integration into the C. reinhardtii chloroplast genome. p322 DNA includes the 5.7-kb region from EcoRI to XhoI in the C. reinhardtii chloroplast genome corresponding to position 44,877–50,577 (www.biology.duke.edu/chlamy_genome/chloro.html). Double-headed arrows indicate regions corresponding to the probes used in the Southern blot analysis. Black boxes indicate, from left to right, psbA exon 5, and the 5S and a small portion of the 23S ribosomal RNA genes, respectively.

Figure 3

Southern and Northern blot analysis of HSV8-lsc in C. reinhardtii chloroplast transformants. (A) C. reinhardtii DNAs were prepared as described in experimental procedures, digested with EcoRI and XhoI, and subjected to Southern blot analysis. Filters were hybridized with the radioactive probes indicated by the double-headed arrows in Fig. 2B. (B) Detection of chloroplast-expressed HSV8-lsc mRNA in transgenic C. reinhardtii strains. Total RNA isolated from untransformed (WT), atpA HSV8-lsc transformed (10-6-3), and rbcL transformed (20-4-4) strains was separated on denaturing agarose gels and blotted to nylon membrane. The membranes were either stained with methylene blue (Bottom) or hybridized with a psbA cDNA probe (Middle) or a hsv8-specific probe (Top).

Figure 4

Expression of HSV8-lsc proteins in bacteria and chloroplast. (A) Twenty micrograms of crude protein from E. coli, WT C. reinhardtii, and transgenic lines 10-6-3 and 20-4-4 were separated by SDS/PAGE and either stained with Coomassie blue (Left) or blotted to nitrocellulose membrane and decorated with an anti-flag antibody (Right). (B) Proteins from E. coli and C. reinhardtii expressing the Hsv8-lsc antibody were separated into soluble and insoluble pellets by centrifugation. Twenty micrograms of protein were either stained with Coomassie blue (Left) or blotted to nitrocellulose membrane and decorated with an anti-flag antibody (Right). (C) Soluble proteins from C. reinhardtii transgenic line 10-6-3 were either treated with (+Bme) or without (no Bme) reducing agent before separation on SDS/PAGE. Proteins were blotted to nitrocellulose membrane and decorated with anti-flag antibody.

Figure 5

Characterization of HSV8-lsc binding to HSV8 viral protein via ELISA. Affinity-purified HSV8-lsc from the transgenic C. reinhardtii strains (10-6-3 and 16-3) were screened in an ELISA assay against HSV proteins prepared from virus-infected cells. A total of 100, 80, 70, 60, 30, 20, 10, or 5 μl of Flag-purified hsv8-lsc were incubated in microtiter plates coated with a constant amount of viral protein. Protein concentrations in these affinity-purified extracts were 13 ng/μl, of which ≈10% was HSV8-lsc as judged by Coomassie staining. Equal volumes of WT C. reinhardtii proteins were used as a negative control (concentration = 1 μg/μl).

Figure 6

Effect of growth condition on accumulation of HSV8-lsc in C. reinhardtii. Before harvest, C. reinhardtii transgenic lines 10-6-3 and 20-4-4 were maintained at either 1 × 10⁶ cells per ml or 1 × 10⁷ cells per ml. Cultures were grown either under continuous illumination or under a 12-h light/12-h dark cycle. Total soluble protein (20 μg) was separated by SDS/PAGE, blotted to nitrocellulose membrane, and decorated with anti-Flag antibody.