New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii

C. reinhardtii strains and culture conditions

The TN72 strain is a psbH::aadA knockout mutant of the cell wall-deficient strain cw15.mt+ (Davies and Plaskitt 1971) and was created as described below. TN72 has been deposited in the publicly available collection of the Chlamydomonas Resource Center (chlamycollection.org) as strain CC-5168. TN72 transformant lines were initially selected on 2 % agar plates containing high salt minimal (HSM) medium but then maintained on Tris-acetate phosphate (TAP) agar plates (Harris et al. 2009) under dim light (5–10 μE/m²/s) at 20 °C. For liquid cultures, cells were grown in TAP medium in conical flasks at 25 °C, 120 rpm at 5 μE/m²/s (for TN72) or 50 μE/m²/s (for transformants). Spectinomycin (100 μg/ml) and erythromycin (10–50 μg/ml) were added to TAP agar medium as required.

Plasmid construction

DNA manipulations were carried out using standard protocols (Green and Sambrook 2012), including transformation of chemically competent E. coli DH5α using ampicillin selection. Oligonucleotide primers were obtained from Eurofins Genomics (Ebersberg, Germany); restriction enzymes and T4 DNA ligase for cloning procedures were purchased from NEB (Ipswich, MA, USA) and Thermo Scientific (Waltham, MA, USA). Details of oligonucleotide primers used for plasmid construction are given in Table S1. Plasmid p72B-aadA-BM, which was used to create TN72, was made by replacing the 0.36-kb MluI-BstXI region of p72B (Bateman and Purton 2000) with the “aadA cassette” (comprising the aadA coding sequence fused to the atpA promoter/5′UTR and rbcL 3′UTR) amplified from pUC-atpX-AAD (Goldschmidt-Clermont 1991) using primers that introduced an MluI and BstXI site at the ends of the cassette. Transformation vector pASapI was created from p72B-SH (Bateman and Purton 2000), by first cutting p72B-SH with SapI (within the pUC8 vector) and EcoRI, end-filling, and religating, thereby removing a 240-bp region of the vector sequence including the unwanted SapI site. Then, the MluI-NcoI region containing the rbcL promoter/5′UTR element was replaced with an equivalent region amplified from atpA to give pASapI in which a SapI site was created immediately downstream of the start codon. Vectors pSRSapI, pPSapI, and pCSapI were created from pASapI by replacing the MluI to SapI region of pASapI with the promoter/5′UTR region of psaA exon 1, psbA, and chlL, respectively. The sequences of pASapI and pSRSapI are given in Fig. S1. The ereB coding sequence was amplified from plasmid pAT72 (Arthur and Courvalin 1986) using primers that introduce an NcoI and SphI site at the start and end of the coding region, respectively, and cloned into these sites in pSRSapI to create pRY129a. The synthetic gene for hGH was codon-optimized for the C. reinhardtii chloroplast using the Codon Usage Database (www.kazusa.or.jp/codon) with SapI and SphI sites added for cloning (Fig. S2). The gene was synthesized by Life Technologies (Carlsbad, CA, USA) and cloned into the four expression vectors.

Chloroplast transformation

Chloroplast transformation was based on a previously described method (Kindle et al. 1991) and involved the agitation of an algal/DNA suspension with glass beads of 400–625 μm diameter. A 400-ml culture grown to early log phase (approx. 2 × 10⁶ cells/ml) was concentrated by centrifugation and resuspended in TAP medium to 4 ml. Three hundred microliters of cells were added to a sterilized 5-ml test tube containing 300 mg sterile glass beads, followed by 5–10 μg circular plasmid DNA. The mixture was agitated vigorously at the maximum speed of a Vortex Genie II (Fisher Scientific, Loughborough, UK) for 15 s. The cells were spread on selective agar plates (TAP + spectinomycin at 100 μg/ml for aadA selection; HSM for psbH selection) after mixing with 0.5 % molten (42 °C) agar of the same selective medium. The plates were incubated at 25 °C in dim light (~2 μE/m²/s) overnight then transferred to a moderate light (~50 μE/m²/s) the next day. Transformant colonies were picked after 2–3 weeks and restreaked to single colonies several times on selective media to ensure homoplasmicity, although this was often achieved after the first streaking. Homoplasmy was determined by PCR using a combination of three primers (Table S1). Total genomic DNA was extracted from a loopful of cells using the Chelex 100 method as described by Werner and Mergenhagen (1998), and PCR amplification was carried out with Phusion DNA polymerase (Thermo Scientific) according to the manufacturer’s instructions. The photosystem II-deficient phenotype of TN72 was confirmed by measuring the photosynthetic capacity through chlorophyll fluorescence (Maxwell and Johnson 2000). Putative transformant lines were spotted onto TAP agar plates, incubated for 5 days and scanned using a pulse-modulated imaging fluorometer (FluorCam 700MF, Photon Systems Instruments, Czech Republic) as described by Wingler et al. (2004).

Western analysis of C. reinhardtii and E. coli transformants

Protein samples from C. reinhardtii were prepared, fractionated on a 15 % acrylamide SDS-PAGE gel and blotted onto nitrocellulose membrane as described by Young and Purton (2014). For immunodetection, membranes were blocked in TBS-T (20 mM Tris, 137 mM NaCl, 1 M HCl pH 7.4, 0.1 % Tween-20) with 0.5 % milk powder overnight at 4 °C. Membranes were incubated with primary antibody in TBS-T with 0.5 % milk (1 h at RT), washed 3 × 5 min in TBS-T, incubated with secondary antibody in TBS-T with 0.5 % milk (1 h at RT), and washed as before. The probed blots were scanned and quantified using an Odyssey Infrared Imaging System (LI-COR). Alternatively, for Fig. 5a, the antibody binding was detected by enhanced chemiluminescence (ECL) using SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific) and Hyperfilm ECL (GE Healthcare). For hGH detection, the primary antibody was chicken polyclonal antibody to human growth hormone (Abcam product ab27223) at 1:5000 dilution and the secondary was donkey anti-chicken IgY IRDye 800CW (LI-COR) at 1:5000, or goat anti-chicken IgY HRP-linked antibody (Abcam product ab97135) at 1:5000 for ECL. For RbcL detection, the primary antibody was anti-RbcL produced in rabbit (a gift from John Gray, Cambridge University, UK) at 1:5000 followed by secondary antibody Goat anti-Rabbit IgG (H&L), Dylight™ 800 Conjugated (Thermo Scientific product 35571) at 1:10,000, or HRP-linked Goat Anti-Rabbit IgG H&L (Abcam product ab6721) at 1:5000 for ECL.

For the detection of proteins in E. coli, 5 ml of an overnight LB culture was harvested by centrifugation (10 min at 4500 g) and resuspended in X/4 ml solution A (0.8 M Tris-HCl pH 8.3, 0.2 M sorbitol, 1 % β-mercaptoethanol) where X is the absorbance of the culture at 600 nm. SDS was added to a final concentration of 1 %. Samples were boiled for 5 min and passed through a 0.5-mm needle then centrifuged (2 min at 21,000 g), and 25 μl of supernatant was loaded. Gel and hGH immunodetection conditions using the ODYSSEY system were as described above. For both the Chlamydomonas and E. coli analysis, pure recombinant hGH (Abcam product 51232) was used as a reference.

Nb2-11 cell proliferation assay for hGH activity

Nb2-11 cells (European Collection of Cell Cultures) were grown in suspension culture in Fischer’s medium supplemented with 10 % fetal bovine serum, 10 % horse serum, 2 mM l-glutamine, 0.075 % sodium bicarbonate, and 0.05 mM β-mercaptoethanol, at 37 °C in an atmosphere containing 5 % CO₂. Cells were starved for 24 h by centrifugation (750 g for 5 min) to remove the culture medium, followed by washing and resuspending the cells in supplemented Fischer’s medium excluding fetal bovine serum. The cells were seeded in a 12-well plate at a concentration of approximately 1 × 10⁵ cells/ml. Recombinant hGH (Abcam) was added to 1 ml of cells in a volume of 5 μl to give a final concentration of 1, 10, or 100 ng/ml. Phosphate buffer (50 mM, pH 7.4) was added to cells as a control and was used to dilute the hGH. Samples were analyzed in triplicate. Cell proliferation was monitored every 24 h using the CASY^® Cell Counter and Analyzer Model TT (Innovatis, Bielefeld, Germany), with cells >8.03 μm being defined as viable. To test C. reinhardtii extracts, transformant TN-hGH, and the negative control TN-C, cells were cultured in 500-ml TAP medium to a concentration of 2 × 10⁶ cells/ml, broken by three cycles of freezing in liquid nitrogen and thawing, centrifuged at 9000 g for 30 min, followed by ultracentrifugation of the supernatant at 100,000 g for 60 min. The hGH content in the TN-hGH extract was quantified by Western blotting as above and a volume containing ~100 ng hGH used in the proliferation assay, together with an equivalent amount of TN-C extract, or phosphate buffer as controls. The experiment was repeated in triplicate with proliferation measured after 96 h.

Sequence data

The Genbank accession number of the synthetic hGH used in the study is KU179444.