Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris

Chuck, Chi Pang; Wong, Chi Lok; Chow, Larry M.C.; Fung, Kwok‐Pui; Waye, Mary Miu Yee; Tsui, Stephen Kwok‐Wing

doi:10.1007/s11262-008-0292-3

Cited by 17 publications

(16 citation statements)

References 18 publications

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“…In agreement with our results, in a previous attempt to express the similar RBD from SARS-CoV-1 Spike in E. coli , this protein was also found in the insoluble fraction, and neither its fusion to thioredoxin, nor to maltose-binding protein, increased its solubility. Moreover, while tagging the protein with glutathione S -transferase (GST) increased its solubility 37 , it remained strongly bound to the bacterial chaperone GroEL even after affinity purification 38 , thus making it unsuitable for downstream applications.…”

Section: Discussionmentioning

confidence: 99%

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

Arbeitman¹,

Auge²,

Blaustein³

et al. 2020

Sci Rep

104

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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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Section: Discussionmentioning

confidence: 99%

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

Arbeitman¹,

Auge²,

Blaustein³

et al. 2020

Sci Rep

104

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“…To overcome specific restrictions to the expression of DENV VLPs, we adopted a well-tolerated P. pastoris yeast cells to produce DENV-2 VLPs. Meanwhile, P. pastoris is one of the most economic and convenient expression systems to produce large amount of antigens for vaccine development [25,26]. It has no pathogenic relationship with man, is free of endotoxin and has been used in industrial fermentations for centuries [20,21].…”

Section: Discussionmentioning

confidence: 99%

Recombinant dengue virus-like particles from Pichia pastoris: efficient production and immunological properties

et al. 2009

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The envelope glycoprotein (E) of flavivirus is the major structural protein on the surface of the mature virions. The complexes of premembrane (prM) and E play important roles in virus assembly and fusion modulation and in potential immunity-inducing vaccines. In the present study, the cDNA encoding prM and E proteins of dengue virus type 2 (DENV-2) was subcloned into the pGAPZalphaA vector and further integrated into the genome of Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. The high-level constitutive expression of recombinant E antigen was achieved in P. pastoris. Both the cell lysate and the culture supernatant, examined by electron microscopy, were found to contain DENV-2 virus-like particles (VLPs) with diameters of about 30 nm. After immunization of BALB/c mice, the VLPs exhibited similar efficacies as inactivated virus in terms of antibody induction and neutralization titer. These results suggest that recombinant DENV VLPs can be efficiently produced in the GAP promoter-based P. pastoris expression system. This system may be useful for the development of effective and economic dengue subunit vaccine.

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“…Pichia pastoris has previously been used for the expression of difficult proteins with high yields, at cheap prices, using scalable protocols (including industries) [ 14 ]. In fact, a domain of the SARS-CoV spike protein was successfully expressed in P. pastoris in 2009 [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

Developing an Optical Interferometric Detection Method based biosensor for detecting specific SARS-CoV-2 immunoglobulins in Serum and Saliva, and their corresponding ELISA correlation

Murillo

Tomé-Amat

Ramírez

et al. 2021

Sensors and Actuators B: Chemical

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The standard rapid approach for the diagnosis of coronavirus disease 2019 (COVID-19) is the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. The detection of specific anti-SARS-CoV-2 immunoglobulins is crucial for screening people who have been exposed to the virus, whether or not they presented symptoms. Recent publications report different methods for the detection of specific IgGs, IgMs, and IgAs against SARS-CoV-2; these methods mainly detect immunoglobulins in the serum using conventional techniques such as rapid lateral flow tests or enzyme-linked immunosorbent assay (ELISA). In this article, we report the production of recombinant SARS-CoV-2 spike protein and the development of a rapid, reliable, cost-effective test, capable of detecting immunoglobulins in serum and saliva samples. This method is based on interferometric optical detection. The results obtained using this method and those obtained using ELISA were compared. Owing to its low cost and simplicity, this test can be used periodically for the early detection, surveillance, detection of immunity, and control of the spread of COVID-19.

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Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris

Cited by 17 publications

References 18 publications

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

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

Recombinant dengue virus-like particles from Pichia pastoris: efficient production and immunological properties

Developing an Optical Interferometric Detection Method based biosensor for detecting specific SARS-CoV-2 immunoglobulins in Serum and Saliva, and their corresponding ELISA correlation

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