Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells
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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 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 T m 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 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.
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Reviewers: Tom Gallagher (Loyola University Chicago) | 📒📒📒 ◻️◻️
Roslyn Bill (Aston University) | 📗📗📗📗◻️ -
Roslyn Bill
Review 2: "Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells"
This pre-print compares S protein RBD production in Pichia pastoris compared to that produced in mammalian cells. While the study is reliable, the manuscript should be revised for accuracy and further functional testing should be performed.
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Tom Gallagher
Review 1: "Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells"
This pre-print compares S protein RBD production in Pichia pastoris compared to that produced in mammalian cells. While the study is reliable, the manuscript should be revised for accuracy and further functional testing should be performed.
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SciScore for 10.1101/2020.09.17.300335: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: STAN No. 4482), under ISO9001 guidelines and those from the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Female mice (6-8 week-old) were immunized intraperitoneally with 40 µg RBD protein produced in P. pastoris in the presence of the HPLC-grade phosphorothioate oligonucleotide CpG-ODN 1826 (5’ TCCATGACGTTCCTGACGTT 3’) (20 µg/mouse/dose) (Oligos etc. Inc. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources RBD expression was confirmed by Western blot analysis using anti-RBD and anti-his antibodies. …SciScore for 10.1101/2020.09.17.300335: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: STAN No. 4482), under ISO9001 guidelines and those from the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Female mice (6-8 week-old) were immunized intraperitoneally with 40 µg RBD protein produced in P. pastoris in the presence of the HPLC-grade phosphorothioate oligonucleotide CpG-ODN 1826 (5’ TCCATGACGTTCCTGACGTT 3’) (20 µg/mouse/dose) (Oligos etc. Inc. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources RBD expression was confirmed by Western blot analysis using anti-RBD and anti-his antibodies. anti-RBDsuggested: Noneanti-hissuggested: NoneExperimental Models: Cell Lines Sentences Resources Expression of RBD was carried out in the HEK-293T cell line kindly provided by Xavier Saelens (VIB-University of Ghent, Belgium). HEK-293Tsuggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)Experimental Models: Organisms/Strains Sentences Resources BALB/c mice were obtained from the animal facility of the Faculty of Veterinary Sciences, University of La Plata (Argentina), and housed at the animal facility of the Instituto de Ciencia y Tecnología Dr. César Milstein, Fundación Pablo Cassará. BALB/csuggested: RRID:IMSR_ORNL:BALB/cRl)Software and Algorithms Sentences Resources Multiple sequence alignment was obtained using MAFFT v.745342. MAFFTsuggested: (MAFFT, RRID:SCR_011811)The RBD region was extracted with the EMBOSS package43 using the RBD region of Uniprot accession QHN73795. EMBOSSsuggested: (EMBOSS, RRID:SCR_008493)Protein identity analysis was performed by BLAST using the RBD protein sequence as a query and an e-value of 0.001 was used. BLASTsuggested: (BLASTX, RRID:SCR_001653)The resulting MS/MS spectra were analyzed using the MASCOT search engine44 (Matrix Science) program and COMET45 at Transproteomic Pipeline. MASCOTsuggested: (Mascot, RRID:SCR_014322)The accessible surface area calculations for the residues of RBD was done using the GetArea server http://curie.utmb.edu/getarea.html using a 1.4 Å probe radius. GetAreasuggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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