key: cord-0800030-vj1xeq6k authors: Berndt, A.; Smalley, T.; Ren, B.; Badary, A.; Sproles, A.; Fields, F.; Torres-Tiji, Y.; Heredia, V.; Mayfield, S. title: Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii date: 2021-01-30 journal: bioRxiv DOI: 10.1101/2021.01.29.428890 sha: 8170391fb8b39fca4583c4e2b82b3e7d7070876e doc_id: 800030 cord_uid: vj1xeq6k Recombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas. RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated, lacking the amino terminus. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms. Since it is known that there are different protein folding and posttranslational modifications 148 made to proteins depending on which organelle a protein is directed to, we generated three 149 versions of the RBD::mClover construct; 1) A chloroplast directed version, produced by adding 150 the psaE chloroplast transit sequence to the N-terminus of the RBD fusion protein, 2) a secreted 151 version, produced by adding Pherophorin 2 (PHC2) signal peptide to the N-terminus of the RBD 152 fusion protein, and 3) a ER-Gogli retained version produced by the addition of a C-terminal 153 KDEL retention motif to the carboxy end of the RBD fusion protein containing the PHC2 154 secretion peptide (Figure 1 B) . 155 156 Transformation and high throughput screening for recombinant protein production 157 The three vectors were linearized and transformed separately into algae via electroporation. 158 Following recovery on complete media, cells were selected on media containing both Zeocin and 159 Hygromycin. Ten days post-transformation, individual colonies were picked into 96-well 160 microtiter plates containing TAP media and grown for two days. The clones were then passaged 161 at a 1:4 dilution in to fresh TAP media for two days and fluorescence analysis, using a plate 162 reader, was used to identify strains with high mClover expression. The mClover fluorescence 163 signal was normalized to chlorophyll fluorescence and compared to both the CC124 starting 164 strain and a previously characterized GFP-expressing strain (Fields, 2019) . Several hundred 165 mClover expressing transformants were recovered for both the secreted and ER retained clones, 166 from three independent transformations, while only a few dozen colonies were recovered from 167 the chloroplast targeted strains, despite the same amount of DNA being used in each of the 168 transformations. Similarly, by mClover fluorescence analysis, 10-30% of all secreted and ER retained transformants showed fluorescence well above wild type, while only about 1-5% of the 170 chloroplast-directed strains showed any mClover fluorescence. The chloroplast localized RBD::mClover protein was then purified using anion exchange 209 chromatography followed by anti-GFP magnetic bead immunoprecipitation, and the partially 210 purified protein products were characterized using protein mass spectrometry analysis. The most 211 N-terminal peptide fragment detectable by mass spectroscopy in the chloroplast-directed 212 RBD::mClover corresponded to a protein product that would be 9 kDa smaller than the predicted 213 51 kDa full length mature protein, while a parallel experiment using the ER-Golgi retained 214 protein identified peptide matches across the entire length of the protein (See Supplemental 215 To characterize the RBD protein produced using C. reinhardtii, we opted to purify the 236 RBD::mClover from the ER-retained version rather the secreted version, because we noticed that 237 the protein had similar molecular mass, suggesting similar post-translational modifications, and 238 because we noticed high molecular weight aggregates in the precipitated secreted proteins. Using 239 a variety of chromatography resins, we were able to purify sufficient amount of ER-retained 240 RBD::mClover to characterize the protein for receptor-interaction activity, and to identify that 241 total RBD::mClover protein was 0.1% of total soluble protein in total cell lysate and 2.6% of 242 total protein after this partial purification. We determined initial and partially purified final Characterization of the same samples using anti-GFP to detect the RBD::mClover gene product. 261 The RBD::mClover can be separated from the lower molecular weight mClover degradation 262 product. 263 The SARS-CoV-2 spike RBD interacts with human host cells through ACE2 receptor binding 266 determined that that HEK cell expressed SARS-CoV-2 RBD C-terminally fused to rabbit IgG Fc 272 (RBD::rFc) could bind to the immobilized ACE2, and could identify that the recombinant RBD 273 bound the ACE2 protein at the EC 50 of ~0.6ng/mL ( Figure 4A ). This affinity is within the nM 274 range previously reported (Liu et al., 2020) but 10-fold higher than the EC 50 reported by the 275 manufacturer (2-6 ng/mL). Next we added a constant amount of our partially purified algae-276 expressed RBD::mClover, and then titrated in the RBD::rFc as a competitor. Using anti-GFP 277 antibodies to detect RBD::mClover bound to the immobilized ACE2 receptor, we showed that 278 the RBD::rFc will compete RBD::mClover binding at an IC 50 of ~1 µg/mL which is 279 approximately equal to the 2 µg/mL of the algae produced RBD::mClover loaded in to each well 280 ( Figure 4 ). This suggests that the algae-produced SARS-CoV-2 RBD is functional in ACE2 281 binding, and appears to have a similar affinity as SARS-CoV-2 RBD produced in HEK cells, the 282 standard for functional SARS-CoV-2 RBD protein. Here we have demonstrated that production of a correctly folded and functional SARS-CoV-2 296 spike protein Receptor Binding Domain is possible in the green alga C. reinhardtii. By fusing 297 the viral protein to a fluorescent mClover protein, we could use a high throughput fluorescent 298 screening strategy to rapidly identify strains of algae expressing sufficient quantities of SARS-299 RBD to test protein accumulation and function. We showed that nuclear encoded transgenes, 300 directed to either the ER or secreted from the cell, produce a fusion protein of the expected size 301 that had the correct amino acid sequence, and that appeared to be correctly folded and post-302 translationally modified, allowing the protein to function in ACE2 receptor binding. Protein 303 targeted to the chloroplast appeared to be truncated, likely by a chloroplast specific protease, 304 resulting in a protein that was not recognized by RBD polyclonal antibodies. Mass spectral 305 analysis suggests that the chloroplast truncated protein is the result of an N-terminal truncation in 306 the chloroplast-directed protein. Using partially purified ER localized algae SARS-CoV-2 RBD 307 to conduct ACE2 receptor binding interaction assays, we could demonstrated that the algae-308 produced ER-retained version of RBD::mClover did indeed interact in a specific manner with its 309 cognate human host receptor, at a perceived affinity similar to mammalian expressed SARS-310 CoV-2 RBD, suggesting that the folding and post translational modifications of the recombinant 311 algae RBD are sufficient to mediate appropriate RBD binding activity. Collectively these data 312 demonstrate the suitability of algae as a platform for the rapid production of recombinant 313 proteins that are correctly folded and post-translationally modified to create a functional protein. 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The Plant 627 Robust 629 Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a 630 Selection Gene and Processing with the FMDV 2A Peptide Production of therapeutic 634 proteins in algae, analysis of expression of seven human proteins in the chloroplast of 635 Chlamydomonas reinhardtii Current advances in the algae-made biopharmaceuticals 639 field Recent 642 advancements in the genetic engineering of microalgae 645 Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: 646 implication for development of RBD protein as a viral attachment inhibitor and vaccine Cryo-EM structure of the 2019-nCoV spike in the prefusion 651 conformation Haycock's blocking solution; 1% wt/vol Polyvinylpyrrolidone in TBSMT (Haycock, 1993) . Protein lysate from the Chloroplast-directed and ER-Golgi Retained strains were prepared as 499 above and RBD protein purified by Anion exchange chromatography using Capto-Q resin. It