Protein Expression and Purification

An effective prophylactic vaccine is urgently needed to protect against SARS-CoV-2 infection. The viral spike, which mediates entry into cells by interacting with the host angiotensin-converting enzyme 2, is the primary target of most vaccines in development. These vaccines aim to elicit protective immunity against the glycoprotein by use of inactivated virus, vector-mediated delivery of the antigen in vivo, or by direct immunization with the purified antigen following expression in a heterologous system. These approaches are mostly dependent on the growth of mammalian or insect cells, which requires a sophisticated infrastructure that is not generally available in developing countries due to the incumbent costs which are prohibitive. Plant-based subunit vaccine production has long been considered as a cheaper alternative, although low expression yields and differences along the secretory pathway to mammalian cells have posed a challenge to producing certain viral glycoproteins. Recent advances that have enabled many of these constraints to be addressed include expressing the requisite human proteins in plants to support the maturation of the protein of interest. In this study we investigated these approaches to support the production of a soluble and putatively trimeric SARS-CoV-2 spike mimetic in Nicotiana benthamiana via transient Agrobacterium-mediated expression. The co-expression of human calreticulin dramatically improved the accumulation of the viral spike, which was barely detectable in the absence of the co-expressed accessory protein. The viral antigen was efficiently processed even in the absence of co-expressed furin, suggesting that processing may have occurred at the secondary cleavage site and was mediated by an endogenous plant protease. In contrast, the spike was not efficiently processed when expressed in mammalian cells as a control, although the co-expression of furin improved processing considerably. This study demonstrates the feasibility of molecular engineering to improve the production of viral glycoproteins in plants, and supports plant-based production of SARS-CoV-2 spike-based vaccines and reagents for serological assays.

The fundamental biology of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.

In this work multiple plasmids have been created to allow the simple Golden Gate cloning of a target gene for recombinant protein production in Escherichia coli. To simplify as much as possible the generation of different target gene vector combinations, the 22 plasmids contain the same Golden Gate cloning sites (BsaI), antibiotic resistance (kanamycin) and promoter (T7) for expression in the standard protein production strain of E. coli BL21[DE3]. The plasmid set includes commonly used tags for purification and assays (his, twin-strep and avi tag) as well as fusion protein partners that may aid target protein solubility and yield, SUMO, MBP, GST and sfGFP. Also included are plasmids with secretion peptide signals for transport of the target protein to the E. coli periplasm via various pathways (SEC, SRP, TatA). We have evaluated the 4 of the vectors using a test super folder GFP insert and found that using the Golden Gate process allows cloning efficiencies of greater than 90% to be routinely obtained. Vectors were further evaluated by expressing and purifying the target insert. The plasmid vector set described herein should prove useful to any investigator who has to routinely evaluate numerous protein expression constructs and are freely available through Addgene.

Nanobodies (NBs) are small antibody fragments derived from camelid heavy-chain antibodies which represent the minimal functional domain capable of antigen recognition and binding. NB are ten times smaller than conventional antibodies, exhibit a compact structure and high stability, making them ideal for recombinant production. The eukaryotic unicellular system Pichia pastoris provides multiple advantages for protein expression, including the ability to perform several eukaryotic posttranslational modifications. In this work, we engineered a modular plasmid sequence that, through specific restriction enzyme cuts and ligations, codes the expression of a secreted anti-mouse kappa chain NB fused with various accessory peptides in P. pastoris. This system enables the incorporation of a plastic binding sequence, a histidine tag (Hisx6) for purification, the horseradish peroxidase (HRP) enzyme for chemiluminescence detection, or the biotinylatable AviTag sequence, in multiple combinations. We successfully expressed and purified anti-kappa NBs fused to a Hisx6-tag ({kappa}NB) and to HRP -Hisx6-tag ({kappa}NB-HRP), with subsequent structural and functional characterization revealing high affinity for mouse immunoglobulins. The {kappa}NB-kappa light chain domain complex was modeled showing a fitted surface interaction of CDR3 domain. The position of a glycan in the complex was modeled predicting that glycan addition would not affect the interaction surface. Accordingly, no functional differences were observed in {kappa}NB after deglycosylation, indicating that high mannose glycan addition has not interfered with its binding capability. Moreover, glycosylated {kappa}NB fused to HRP was expressed with retained HRP activity, and proved to be functional as a secondary antibody, demonstrating the systems versatility in producing NBs and conjugated NBs with posttraslational modification that may be required for diverse biotechnological applications.

The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from SARS-CoV-2 Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 {degrees}C and 52 {degrees}C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by HPLC, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

Heterologous protein production is often required to investigate the structural, biochemical, and biophysical properties of a protein of interest. Frequently, optimisation of expression conditions is required to obtain soluble protein and maximise yield. Trialling a variety of solubility and purification tags, as well as constructs containing different regulatory elements, is desirable. Golden Gate cloning allows modular assembly of different constructs using Type IIS restriction enzymes. The pOPIN vector suite, which utilises the T7 expression system, has been adapted to be compatible with Golden Gate assembly. Here, we present the pOPARA vectors. Expression from pOPARA vectors is driven by the araBAD promoter (pBAD) and is induced by addition of arabinose to the culture medium. pOPARA allows modular assembly of expression constructs using Golden Gate cloning with the CDS of interest and an optional C-terminal tag. pOPARA1 contains a carbenicillin resistance cassette flanked by restriction sites to allow exchange of the selectable markers. In pOPARA2, the carbenicillin resistance cassette has been exchanged for a spectinomycin resistance cassette. We demonstrate that both vectors can be used to express and produce a control protein.

The aqueous extract of Cynara cardunculus flowers is traditionally used in cheese production across Mediterranean countries. To meet the growing industrial demand for plant-based milk-clotting enzymes and to explore potential biotechnological applications, we initiated a study to heterologously produce cyprosin B (CYPB), a key milk-clotting enzyme from C. cardunculus, in Nicotiana benthamiana. We also investigated the role of its plant-specific insert (PSI) domain in the CYPBs activity and its localization. In this study, full-length CYPB and a PSI domain deleted CYPB (CYPB{Delta}PSI) were transiently expressed in N. benthamiana leaves using Agrobacterium-mediated infiltration. The leaves were harvested nine days post-infiltration, and proteins were purified, yielding approximately 81 mg/kg (CYPB) and 60 mg/kg (CYPB{Delta}PSI) fresh weight. CYPB{Delta}PSI showed significantly higher proteolytic activity (156.72 IU/mg) than CYPB (57.2 IU/mg), indicating that the PSI domain is not essential for enzymatic activity and that its removal results in enhanced enzymatic efficiency. In the milk-clotting activity assay, CYPB{Delta}PSI demonstrated a significantly faster clotting time than full-length CYPB, indicating enhanced milk-clotting efficiency for CYPB{Delta}PSI. Subcellular localization studies revealed that CYPB and PSI were localized in the vacuole and endocytic vesicles. In contrast, CYPB{Delta}PSI was primarily localized in the endoplasmic reticulum (ER) and the tonoplast, suggesting that the PSI domain is critical for vacuolar targeting and membrane permeabilization that affects overall protein yield. This study demonstrates the feasibility of using N. benthamiana as a platform for the scalable production of more efficient recombinant CYPB. It highlights the multifunctional role of the PSI domain in vacuolar sorting without impairing its functionality. These results underscore the potential of plant-based expression systems as a viable alternative for the industrial production of plant milk-clotting enzymes, with significant implications for sustainable cheese production.

High-throughput screening (HTS) of proteins has a wide range of applications across the biology, biotechnology, and medicine disciplines. These include yield optimisation, drug or biomarker discovery, and protein engineering, among others. Factors that need to be considered in designing high throughput protein expression and screening methods, be that for expression, activity, stability, or binding as says, include the required yield, reproducibility, solubility, stability, purity and activity of the protein. Thus, larger culture volumes and time-consuming manual protein extraction and purification steps are normally required to produce sufficient quantity of protein of appropriate purity. This limits the type of assay, and number of protein variants that can be simultaneously tested in an experiment. Here we describe a HTS protocol that allows the overnight expression, export and assay of recombinant proteins from E. coli cells in the same multi-well plate tube. The protocol uses a recently described Vesicle Nucleating peptide (VNp) technology that promotes high yield vesicular export of functional proteins from E. coli into the culture media. The resulting protein is of sufficient purity and yield that in can be used directly in plate-based enzymatic assays without additional purification. This simple single plate protocol allows itself to a wide range of high-throughput research and development screening applications, ranging from streamlining protein production and identification of activity enhancing mutations, to ligand screening for basic research, biotechnological and drug discovery applications.

The SARS-CoV-2 coronavirus causes severe acute respiratory syndrome and has caused a global pandemic by causing the COVID-19 disease. To monitor and control it, diagnostic methods such as molecular and serological tests are necessary. The serological approach uses SARS-CoV-2 antigens to detect the antibodies present in patients using quantitative techniques such as enzyme-linked immunosorbent assay (ELISA) or qualitative rapid tests such as lateral flow chromatography (RDTs). The main antigens used are the spike protein (S) and the nucleocapsid protein (N). Both proteins are obtained in different expression systems, in eukaryotic cells, their production is expensive, so in this work we chose a simpler and cheaper system such as prokaryotic to express and purify the N protein. Thereore, the nucleotide sequence had to being optimized to be expressed in Escherichia coli. The protein N is sensitive to E.coli proteases and also has the ability to self-proteolyze under native conditions, degrading into different fragments. However, under denaturing conditions, using urea and at pH 5.3 it is stable and efficiently purified using metal exchange chromatography (IMAC). In our purification strategy, we surprisingly found that by not using a sonicator, a homogeneous and time-stable preparation of the recombinant antigen is obtained. An approximate yield of 200 mg / L was obtained. It was then tested with healthy sera and sera from COVID-19 convalescent patients in Wester-blot tests that were able to recognize it. Our work provides a novel strategy to produce the SARS-CoV-2 protein N so that it can be used as an input in the development and innovation of serological tests in the diagnosis of COVID-19.

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.

Baculovirus technology has been the most commonly used expression system for insect cells both due to its potential to generate a large amount of recombinant protein as well as the benefit of post-translational modifications. The most commonly used system to generate recombinant baculoviruses is the Tn7 transposition-based technology known as Bac-to-Bac. Although improvements have been made to this system to further improve quality and reproducibility of baculovirus production, recent data suggests that improved strains still have potential issues with contamination of non-recombinant baculovirus caused by improper transposition into a Tn7 site in the E. coli chromosome. Here we describe a new option for alteration of the E. coli genome to completely block the native Tn7 attachment site, leading to far fewer false positive bacmid colonies being selected and eliminating all risk of non-recombinant baculovirus production.

The SLAPTAG is a novel molecular TAG derived from a protein domain present in the sequence of Lactobacillus acidophilus SlpA (SlpA284-444). Proteins from different biological sources, with different molecular weights or biochemical functions, can be fused in frame to the SLAPTAG and efficiently purified by the specific binding to a bacterial-derived chromatographic matrix named here Bio-Matrix (BM). Different binding and elution conditions were evaluated to set an optimized protocol for the SLAPTAG-based affinity chromatography (SAC). The binding equilibrium between SLAPTAG and BM was reached after a few minutes at 4{degrees}C, being the apparent dissociation constant (KD) of 4.3 {micro}M, a value which is similar to different Kd determined for other S-layer proteins and their respective bacterial cell walls. A reporter protein was generated (H6-GFP-SLAPTAG) to compare the efficiency of the SAC against a commercial system based on a Ni2+-charged agarose matrix, observing no differences in the H6-GFP-SLAPTAG purification performance. The stability and reusability of the BM were evaluated, and it was determined that the matrix was stable for more than a year, being possible to reuse it five times without a significant loss in the efficiency for protein purification. Alternatively, we explored the recovery of bound SLAP-tagged proteins by proteolysis using the SLAPASE (a SLAP-tagged version of the HRV-3c protease) that released a tag-less GFP (SLAPTAG-less). Additionally, iron nanoparticles were linked to the BM and the resulting BMmag was successfully adapted for a magnetic SAC, a technique that can be potentially applied for high-throughput-out protein production and purification.

Phage display links phenotype of displayed polypeptides with DNA sequence in phage genome and offers a universal method for discovery of proteins with novel properties. Injection of phage-displayed libraries in living organisms further provides a unique and powerful approach to optimize biochemical, pharmacological and biological properties of the displayed peptides, antibodies and other proteins in vivo. However, over 60% of the proteome is comprised of multi-domain proteins, and display of large multi-subunit proteins on phages remains a challenge. Majority of protein display systems are based on monovalent phagemid constructs but methods for robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a [~]200 kDa tetrameric protein tetrameric L-asparaginase on M13 phage produced by ligation of SpyCatcher-Asparaginase fusion (ScA) to prospectively barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 minor coat protein or p8 major coat protein yielded constructs with five copies of ScA displayed on p3 (ScA5-phage) and 50 copies of ScA on p8 protein (ScA50-phage). ScA remained active after conjugation. It could be easily produced directly from lysates of bacteria that express ScA. Display constructs of different valency can be injected into mice and analyzed by deep-sequencing of the DNA barcodes associated phage clones. In these multiplexed studies, we observed a density-dependent clearance rate in vivo. A known clearance mechanism of L-asparaginase is endocytosis by phagocytic cells. Our observations, thus, link the increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo. In conclusion, we demonstrate that a multivalent display of L-asparaginase on phage could be used to study the circulation life of this protein in vivo and such approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multi-subunit proteins in vivo. Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/432100v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@18edf0org.highwire.dtl.DTLVardef@ad55c6org.highwire.dtl.DTLVardef@1da47d8org.highwire.dtl.DTLVardef@1a2ec32_HPS_FORMAT_FIGEXP M_FIG C_FIG

Many protein expression and purification systems are commercially available to provide a sufficient amount of pure, soluble and active native protein, such as the pMAL system based on E. coli maltose binding protein tag (MBP). Adding specific amino acid tags to the N- or C-terminus of the protein increases solubility and facilitates affinity purification of proteins. However, many of expressed tagged proteins consequently lose functionality, particularly small peptides such as antimicrobial peptides (AMPs). Objective of this study was to redesign the pMAL expression vector in order to increase the efficacy of MBP tag separation from native peptides. Redesign of the pMAL expression vector included introduction of the His6 tag and the enterokinase cleavage site downstream from the original MBP tag and Xa cleavage site enabling purification of native and active peptide (P) following two-step affinity chromatography. In the first step the entire MBP-His6-P fusion protein is purified through binding to Ni-NTA agarose. In the second step, the purification was performed by adding mixture of amylose and Ni-NTA agarose resins following cleavage of the fusion protein with active His6 tagged enterokinase. This removes MBP-His6 and His6-enterokinase leaving pure native protein in solution. The redesigned pMAL vectors were optimized for cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides expression. Two-step purification protocol was successfully applied in purification of active native AMPs, lactococcin A and human {beta}-defensin. Taken together, we established the optimal conditions and pipeline for overexpression and purification of large amount of native peptides, that can be implemented in any laboratory.

The spike protein is the major protein on the surface of coronaviruses. It is therefore the prominent target of neutralizing antibodies and consequently the antigen of all currently admitted vaccines against SARS-CoV-2. Since it is a 1273-amino acids glycoprotein with 22 N-linked glycans, the production of functional, full-length spike protein was limited to mammalian and insect cells, requiring complex culture media. Here we report the production of full-length SARS-CoV-2 spike protein - lacking the C-terminal membrane anchor - as a secreted protein in the prefusion-stabilized conformation in the unicellular green alga Chlamydomonas reinhardtii. We show that the spike protein is efficiently cleaved at the furin cleavage site during synthesis in the alga and that cleavage is abolished upon mutation of the multi-basic cleavage site. We could enrich the spike protein from culture medium by ammonium sulfate precipitation and demonstrate its functionality based on its interaction with recombinant ACE2 and ACE2 expressed on human 293T cells. Chlamydomonas reinhardtii is a GRAS organism that can be cultivated at low cost in simple media at a large scale, making it an attractive production platform for recombinant spike protein and other biopharmaceuticals in low-income countries.

The Ebola virus (EBOV) disease has caused serious and recurrent epidemics in recent years, resulting in a fatality rate of nearly 50%. The most effective experimental therapy against the EBOV is the use of monoclonal antibodies (mAbs). In this work, we describe the development of HEK293T cells engineered for the transient and stable expression of mAb13C6, a neutralizing anti-EBOV monoclonal antibody. We transfected the HEK293T cells with a tricistronic vector to produce the heavy and the light chain of the antibody 13C6 and intracellular Green Fluorescent Protein (GFP) using Lipofectamine 3000. We then selected the transfected cells using puromycin pressure, dilution cloning, and cloning disks. This integrated strategy generated mAb-producing cells in 7 days with a transient expression of [~]1 mg/L. Stable pools were produced after 4 weeks, with expression levels of [~]0.8 mg/L. Stable clones with expression levels of [~]1.8 mg/L were obtained within 10 weeks. The produced antibodies exhibited the expected functionality; they recognized the GP glycoprotein of the Ebola virus in both ELISA assays and cell binding experiments using HEK293T cells engineered to express the EBOV GP at their membrane surface. By the combined use of GFP and the set of selection techniques here described, we drastically reduced the time from transfection to stable clone generation without resorting to costly equipment. In outbreaks or emergencies, this platform can significantly shorten the development of new biopharmaceuticals and vaccines.

Diverse developmental signals and pro-death stresses converge on regulation of the mitochondrial pathway of apoptosis. BAX, a pro-apoptotic BCL-2 effector, directly forms proteolipid pores in the outer mitochondrial member to activate the mitochondrial pathway of apoptosis. BAX is a viable pharmacological target for various human diseases, and increasing efforts have been made to study the molecular regulation of BAX and identify small molecules selectively targeting BAX. However, generating large quantities of monomeric and functionally-competent BAX has been challenging due to its aggregation-prone nature. Additionally, there is a lack of detailed and instructional protocols available for investigators who are not already familiar with recombinant BAX production. Here, we present a comprehensive high-yield protocol for expressing, purifying, and storing functional recombinant BAX protein. We utilize an intein-tagged BAX construct and employ a two-step chromatography strategy to capture and purify BAX, and provide example standard assays to observe BAX activation. We also highlight best practices for handling and storing BAX to effectively preserve its quality, shelf-life, and function.

Death domains play a crucial role in signaling pathways related to inflammation and programmed cell death, rendering them promising targets for therapeutic interventions. However, their expression as recombinant proteins often pose challenges. Here, we present expression screening of TNFR1, IRAK4, and MyD88 death domains in E. coli, followed by the biophysical characterization of TNFR1 death domain after subsequent construct optimization. The study also discusses the influence of pH and ionic strength on TNFR1R347A stability, providing statistical models to predict optimal conditions of the buffer to achieve the highest protein stability. HighlightsO_LIOptimization of expression conditions for TNFR1R347A, MyD88, IRAK4 death domains in E. coli BL21(DE3) cells. C_LIO_LIHigh-yield production of soluble monomeric TNFR1R347A death domain. C_LI

Eukaryotic cell-free protein synthesis (CFPS) systems have the potential to simplify and speed up the expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and the inability to scale such systems have so far prevented their widespread adoption in protein research and manufacturing. Here, we present a detailed demonstration for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in under 48 hours, complete with native disulfide bonds and N-glycosylation. An optimised version of the technology is commercialised as ALiCE(R), engineered for high yields of up to 3 mg/mL. Recent advances in the scaling of BYL production methodologies have allowed scaling of the CFPS reaction. We show simple, linear scale-up of batch mode reporter proten expression from a 100 L microtiter plate format to 10 mL and 100 mL volumes in standard Erlenmeyer flasks, culminating in preliminary data from 1 L reactions in a CELL-tainer(R) CT20 rocking motion bioreactor. As such, these works represent the first published example of a eukaryotic CFPS reaction scaled past the 10 mL level by several orders of magnitude. We show the ability of BYL to produce the simple reporter protein eYFP and large, multimeric virus-like particles directly in the cytosolic fraction. Complex proteins are processed using the native microsomes of BYL and functional expression of multiple classes of complex, difficult-to-express proteins is demonstrated, specifically: a dimeric, glycoprotein enzyme, glucose oxidase; the monoclonal antibody adalimumab; the SARS-Cov-2 receptor-binding domain; human epidermal growth factor; and a G protein-coupled receptor membrane protein, cannabinoid receptor type 2. Functional binding and activity are shown using a combination of surface plasmon resonance techniques, a serology-based ELISA method and a G protein activation assay. Finally, in-depth post-translational modification (PTM) characterisation of purified proteins through disulfide bond and N-glycan analysis is also revealed - previously difficult in the eukaryotic CFPS space due to limitations in reaction volumes and yields. Taken together, BYL provides a real opportunity for screening of complex proteins at the microscale with subsequent amplification to manufacturing-ready levels using off-the-shelf protocols. This end-to-end platform suggests the potential to significantly reduce cost and the time-to-market for high value proteins and biologics.

Serology testing for COVID-19 is highly attractive because of the relatively short diagnosis time and the ability to test for an active immune response against the SARS-CoV-2. In many types of serology tests, the sensitivity and the specificity are directly influenced by the quality of the antigens manufactured. Protein purification of these recombinantly expressed viral antigens [e.g., spike and its receptor binding domain (RBD)] is an important step in the manufacturing process. Simple and high-capacity protein purification schemes for spike, RBD, and CR3022 mAb, recombinantly expressed in CHO and HEK293 cells, are reported in this article. The schemes consist of an affinity chromatography step and a desalting step. Purified proteins were validated in ELISA-based serological tests. Interestingly, extracellular matrix proteins [most notably heparan sulfate proteoglycan (HSPG)] were co-purified from spike-expressing CHO culture with a long cultivation time. HSPG-spike interaction could play a functional role in the pathology and the pathogenesis of SARS-CoV-2 and other coronaviruses.