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Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells

Argentinian AntiCovid Consortium. Sci Rep. .

Abstract

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 severe acute respiratory syndrome coronavirus-2 (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 °C and 52 °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 high-performance liquid chromatography, 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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Structure of SARS-CoV-2 Receptor Binding Domain bound to ACE2. The secondary structure elements of RBD are differentially colored (Alpha helices: purple, 3_10 helices: iceblue, beta strands: yellow, and turns/coil: cyan). Disulfide bridges (red) and tryptophan residues (blue) are shown as sticks, while N-glycosylation asparagine residues (green) are shown as VDW spheres. The region of ACE2 encompassing residues 1–115 (colored white) which interacts with RBD is also shown. The structure was generated using Protein Data Bank (PDB) structures 6xm0 and 6m0j.
Figure 2
Figure 2
Analysis by SDS-PAGE and Reverse Phase (RP)-HPLC of RBD produced in P. pastoris or HEK-293T, and purified by NTA-Ni2+. Analysis of recombinant RBD fractions eluted from a NTA-Ni2+ column by 300 mM imidazole after purification from supernatants of a P. pastoris culture (A) or of HEK-293T cells (B). (C) RP-HPLC Analysis of RBD. Profiles for RBD produced in P. pastoris (red) and HEK-293T mammalian cells (black). The inset shows the expanded region of the chromatogram where the highest peaks eluted. The dashed blue line indicates the variation of acetonitrile (% v/v) during the experiments. Peaks 1, 2 and 3 from RBD produced in P. pastoris correspond to areas of 10.1, 2.8 and 87.1%, respectively.
Figure 3
Figure 3
Analysis of the glycosylation status of RBD produced in HEK-293T and P. pastoris. (A) Schematic representation of SARS-CoV-2 S glycoprotein. N-terminal domain (NTD), receptor-binding domain (RBD), furin cleavage site (S1/S2), central helix (CH), connector domain (CD), and transmembrane domain (TM) are displayed. Residues involved in RBD glycosylation are shown (O- and N-glycosylations are indicated by dotted and solid lines, respectively). (B) Endoglycanase treatment of RBD. Purified RBD (3 µg) from mammalian or yeast culture supernatants was denatured 10 min at 100 °C and digested with PNGase F (500 mU) or EndoH (5 mU) during 2 h at 37 °C. Proteins were separated in a 14% SDS-PAGE gel. The positions of non-glycosylated and glycosylated RBD isoforms are indicated. The bands corresponding to PNGaseF (36 KDa) and EndoH (29 KDa) are indicated by empty or full arrowheads, respectively.
Figure 4
Figure 4
MALDI TOF Spectra for RBD Samples. (A) RBD prepared in HEK-293T and (B) RBD prepared in P. pastoris. As expected, the glycosylated species from P. pastoris have a broad mass. 1Cys is oxidized, 2Calculated without glycosylation.
Figure 5
Figure 5
Absorption spectroscopy. (A) Spectra corresponding to purified RBD produced in HEK-293T cells (black), P. pastoris clones 1 (blue) and 7 (red), and a simulated RBD spectrum (green) (27.5 μM) calculated from its composition of aromatic amino acid (15 Phe, 15 Tyr, and 2 Trp in RBD). (B) Fourth derivative spectra corresponding to the aromatic amino acids (Tyr (blue), Trp (violet), Phe (yellow)) and a simulated RBD spectrum (green). (C) Comparison between the fourth derivative spectra from RBD obtained in HEK-293T (black), P. pastoris RBD Clone 7 (red) and the simulated spectrum presented in A (green).
Figure 6
Figure 6
Hydrodynamic behavior of RBD. (A) SEC-HPLC of RBD produced in P. pastoris (red), HEK-293T (black) and molecular weight markers (dashed green line). This analysis was carried out by injecting 50 μL protein aliquots (0.70 and 0.75 mg/mL for RBD produced in P. pastoris and HEK-293T, respectively) in 20 mM Tris–HCl, 100 mM NaCl, pH 7.0 buffer. The inset shows the correlation between molecular weight and elution volume obtained from the molecular weight markers: (1) gammaglobulin (158 kDa), (2) ovoalbumin (44 kDa), (3) myoglobin (17 kDa), and (4) vitamin B12 (1350 Da). (B) Deconvolution analysis of the chromatographic profile from RBD from P. pastoris. The experimental profile (red), deconvolution of the peak in two different gaussian curves (green and yellow) and the sum of the deconvoluted peaks (blue) are compared.
Figure 7
Figure 7
Conformation and stability of different purified RBD forms characterized by circular dichroism (CD) Spectroscopy, Tryptophan Fluorescence and Temperature-induced Denaturation. (A) Far-UV CD spectra of RBD produced in HEK-293T cells (black), and two different preparations of RBD produced in P. pastoris (red and blue). (B) Tryptophan fluorescence emission was monitored by excitation at 295 nm in 20 mM Tris–HCl, 100 mM NaCl, pH 7.0 at 25 °C. The spectra of RBD obtained in HEK-293 T (black) and in P. pastoris (red) are shown in native conditions (solid line) and in the presence of 4.0 M GdmCl (dashed line) after a 3 h incubation. Refolding of RBD produced in P. pastoris was performed by dilution to final concentrations of 0.7 M (red dot line) and 1.0 M (red dash-dot line) GdmCl. (C) Stability analysis of RBD. Temperature-induced denaturation of RBD produced in P. pastoris (red) and HEK-293T cells (black) under different ionic strength conditions (75, 150, 300 and 500 mM NaCl) was followed by Sypro-orange fluorescence.
Figure 8
Figure 8
Subdomains and distribution of residue types on RBD. The Core (gray) and the RBM (green) regions are shown. Panels A, B, and C, shows the non-polar residues (orange: A, C, G, I, L, M, F, P, W and V), polar (violet: N, Q, S, T and Y), and charged residues (blue: basic K, R and H, red: acid D and E), respectively. To build the models we used the chain E of pdb structure 6m0j.
Figure 9
Figure 9
Evaluation of the cross-reactivity of antibodies produced in mice immunized with P. pastoris RBD. (A) Titers of antibodies obtained by immunization with RBD from P. pastoris plus adjuvants. Each bar represents the group mean (n = 5) for specific titers as determined by end-point-dilution ELISA. ELISA was performed with plates coated with RBD protein produced in HEK-293T cells (black sparse bars) or P. pastoris (red sparse bars). First dose corresponds to blood samples obtained 30 days post-first immunization, and second dose to samples obtained 20 days post-second immunization. Pre IS, Pre Immune Sera; RBD + Adjuvants, RBD produced in P. pastoris + Al(OH)3 + CpG-ODN 1826; Control, Al(OH)3 + CpG-ODN 1826. P values indicate significant differences between different groups. Bars indicate SD. P values (t-test) are shown for statistically significant differences (p < 0.05). (B) Purified RBD produced in HEK-293T (1.0 μg) and in P. pastoris (3.0 μg) were analyzed by Western blot using sera from mice immunized with RBD produced in HEK-293T (anti-RBD HEK-293T, left), or in P. pastoris (anti-RBD, P. pastoris center). As a control a primary antibody against the His tag present in both RBD recombinant proteins was used (right).
Figure 10
Figure 10
P. pastoris biomass concentration (g DCW/L) evolution during bioreactor fermentation. P1: batch phase in LSBM glycerol 40 g/L, P2: Fed-batch phase with 600 g/L glycerol solution, P3: Adaptation phase with glycerol (600 g/L):methanol (3:1) mixture, P4: Induction phase with methanol as the sole carbon source. Error bars indicate 2SD.
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