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Carsten Watzl - One of the best experts on this subject based on the ideXlab platform.
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Current Protocols in Protein Science - Production and use of trimeric isoleucine zipper fusion proteins to study surface Receptor ligand interactions.
Current Protocols in Protein Science, 2006Co-Authors: Carsten WatzlAbstract:Soluble Recombinant forms of surface Receptors are commonly used to study the interaction between cell surface Receptors and their ligands. By fusing the extracellular domain of the Receptor to an isoleucine zipper (ILZ) sequence, it is possible to generate Recombinant Receptor fusion proteins that form trimers in solution. These ILZ fusion proteins demonstrate several advantages over the commonly used Fc fusion proteins. This unit describes the production and purification of Recombinant ILZ fusion proteins. A protocol is also provided for the use of ILZ fusion proteins to study cell surface Receptor-ligand interactions on intact cells by flow cytometry. Keywords: surface Receptor; soluble Receptor fusion protein; Recombinant protein; Receptor-ligand interaction; trimer
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Production and use of trimeric isoleucine zipper fusion proteins to study surface Receptor ligand interactions.
Current protocols in protein science, 2006Co-Authors: Carsten WatzlAbstract:Soluble Recombinant forms of surface Receptors are commonly used to study the interaction between cell surface Receptors and their ligands. By fusing the extracellular domain of the Receptor to an isoleucine zipper (ILZ) sequence, it is possible to generate Recombinant Receptor fusion proteins that form trimers in solution. These ILZ fusion proteins demonstrate several advantages over the commonly used Fc fusion proteins. This unit describes the production and purification of Recombinant ILZ fusion proteins. A protocol is also provided for the use of ILZ fusion proteins to study cell surface Receptor-ligand interactions on intact cells by flow cytometry.
Hartmut Michel - One of the best experts on this subject based on the ideXlab platform.
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heterologous expression and comparative characterization of the human neuromedin u subtype ii Receptor using the methylotrophic yeast pichia pastoris and mammalian cells
The International Journal of Biochemistry & Cell Biology, 2007Co-Authors: Arun K. Shukla, Christoph Reinhart, Winfried Haase, Hartmut MichelAbstract:Abstract Neuromedin U (a neuropeptide) plays regulatory roles in feeding, anxiety, smooth muscle contraction, blood flow and pain. The physiological actions of NmU are mediated via two recently identified G protein-coupled Receptors namely the neuromedin U type 1 Receptor (NmU1R) and the neuromedin U type 2 Receptor (NmU2R). Despite their crucial roles in cell physiology, structural information on these Receptors is limited, mainly due to their low expression levels in native tissues. Here, we report the overexpression of the human NmU2R in the methylotrophic yeast Pichia pastoris and baby hamster kidney (BHK) cells using the Semliki Forest virus (SFV) system. The Recombinant Receptor was expressed as a fusion protein with three different affinity tags namely, the Flag tag, the histidine 10 tag and the biotinylation domain of Propionobacterium shermanii. Expression level of the Recombinant Receptor was 6–9 pmol/mg under optimized conditions, which is significantly higher than the expression level in the native tissues. The Recombinant Receptor binds to its endogenous ligand neuromedin U with high affinity (Kd = 0.8–1.0 nM) and the binding constant for the Recombinant Receptor is similar to that of the wild type NmU2R. Enzymatic deglycosylation suggested that the Recombinant NmU2R was glycosylated in P. pastoris, but not in BHK cells. Confocal laser scanning microscopy and immunogold labelling experiment revealed that the Recombinant Receptor was predominantly localized in the intracellular membranes. To our knowledge, this is the first report of heterologous overexpression of an affinity tagged Recombinant NmU2R and it should facilitate further characterization of this Receptor.
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Functional overexpression and characterization of human bradykinin subtype 2 Receptor in insect cells using the baculovirus system.
Journal of cellular biochemistry, 2006Co-Authors: Arun K. Shukla, Christoph Reinhart, Winfried Haase, Hartmut MichelAbstract:Bradykinin exerts its actions via binding to B1 and B2 Receptors (B1R and B2R), which are members of G protein-coupled Receptor superfamily. B2R is constitutively expressed in a variety of cells such as endothelial cells, vascular smooth muscle cells, and cardiomyocytes and it is an important drug target for the treatment of cardiovascular disorders. During this study, the human B2R was functionally overexpressed in insect cells using the baculovirus expression system. The maximum expression level in Sf9 cells under optimized condition was 10 pmol/mg. This corresponds to approximately 0.25 mg active Receptor per liter culture. The Recombinant Receptor showed high affinity for its endogenous ligand bradykinin, similar to the B2R expressed in native tissues. Functional coupling of the Recombinant Receptor to the endogenous Gαs protein was demonstrated via cAMP release assay upon agonist stimulation. Confocal laser scanning microscopy and immunogold-labeling experiment revealed that the Recombinant B2R was mainly localized intracellularly and only a minor fraction of the Recombinant Receptor reached the plasma membrane. To our knowledge, this is the first report of high level expression of Recombinant B2R in insect cells and provides a way for large scale production and structural characterization of this Receptor. J. Cell. Biochem. 99: 868–877, 2006. © 2006 Wiley-Liss, Inc.
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comparative analysis of the human angiotensin ii type 1a Receptor heterologously produced in insect cells and mammalian cells
Biochemical and Biophysical Research Communications, 2006Co-Authors: Arun K. Shukla, Christoph Reinhart, Hartmut MichelAbstract:Abstract Angiotensin II type 1a Receptor (AT 1a R) is a member of GPCR superfamily and it plays crucial roles in the regulation of blood pressure, hormone secretion and renal functions. Here, we report functional overexpression and characterization of the human AT 1a R in insect cells using the baculovirus system and in mammalian cells using the Semliki Forest virus system. The Recombinant Receptor was expressed at a level of 29–32 pmol/mg and it binds to angiontensin II with high affinity ( K d = 0.98–1.1 nM). Angiotensin II stimulated accumulation of inositol phosphate and phosphorylation of MAP kinase was also observed, which indicated that the Recombinant AT 1a R could couple to endogenous Gα q protein. Confocal laser scanning microscopy revealed that the Recombinant Receptor was predominantly localized in the plasma membrane and agonist induced internalization of the Recombinant AT 1a R was also observed. The Recombinant AT 1a R was expressed in glycosylated form and in vivo inhibition of glycosylation suppressed its surface expression.
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biochemical and pharmacological characterization of the human bradykinin subtype 2 Receptor produced in mammalian cells using the semliki forest virus system
Biological Chemistry, 2006Co-Authors: Arun K. Shukla, Christoph Reinhart, Winfried Haase, Hartmut MichelAbstract:Bradykinin, a vasoactive peptide, plays a crucial role in many cardiovascular processes via activation of the bradykinin subtype 2 Receptor (B2R). B2R, a member of the G protein-coupled Receptor (GPCR) superfamily, is a potential drug target in the treatment of cardiovascular disorders, pain and inflammation. In this study, human B2R was expressed at high levels in baby hamster kidney (BHK) cells using Semliki Forest virus-based vectors. The Recombinant Receptor was produced as a fusion protein with affinity tags and an expression level of 11 pmol/mg (i.e., approx. 0.2 mg of active Receptor per liter of culture) was obtained. Radioligand binding analysis revealed that the Recombinant Receptor binds to its endogenous ligand bradykinin with high affinity (Kd = 0.12 nM) and its pharmacological profile was similar to that of B2R in native tissues. Bradykinin-stimulated accumulation of inositol phosphate was observed in BHK cells expressing the Recombinant Receptor, which indicated the activation of endogenous G alpha(q) protein by the Recombinant B2R. Confocal laser scanning microscopy and immunogold staining revealed that the Recombinant Receptor was predominantly localized intracellularly. To the best of our knowledge, this is the first report of an affinity-tagged Recombinant B2R been expressed at high levels in BHK cells and extensively characterized.
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solubilization and purification of the human etb endothelin Receptor produced by high level fermentation in pichia pastoris
Receptors & Channels, 2001Co-Authors: Hilmar Schiller, Hartmut Michel, Eva Molsberger, Paul Janssen, Helmut ReilanderAbstract:In the present report, the successful solubilization and purification of the ETB Receptor heterologously produced in the methylotrophic yeast P. pastoris is described for the first time. In comparison to the baculovirus system where successful production, solubilization and purification have already been reported, handling and up-scaling of Recombinant P. pastoris cells was much easier and less time consuming. Recombinant P. pastoris clones producing two different ETB Receptor constructs were grown in a fermenter to a density of about 360 g/l. After induction with methanol, a production level of maximally 45 pmol/mg was obtained, a value which is in the range of that reported for baculovirus-infected insect cells. A method for the large-scale preparation of membranes was established. Solubilization of the Recombinant ETB Receptor was achieved with the detergent n-dodecyl-/beta-D-maltopyranoside. The stability of the solubilized and ligand-bound Receptor was examined in detail. Subsequently, two purification methods for two different Receptor constructs were tested and a large-scale procedure for isolation of Recombinant Receptor was established. In general, the purification methods described herein will be adaptable to other G protein-coupled Receptors heterologously produced in heterologous expression systems including P. pastoris.
Arun K. Shukla - One of the best experts on this subject based on the ideXlab platform.
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heterologous expression and characterization of the Recombinant bradykinin b2 Receptor using the methylotrophic yeast pichia pastoris
Protein Expression and Purification, 2007Co-Authors: Arun K. Shukla, Winfried Haase, Christoph Reinhart, Hartmut MichelAbstract:Abstract The human bradykinin subtype 2 Receptor (B 2 R), a member of class A GPCRs, was heterologously expressed in the methylotrophic yeast Pichia pastoris . The Recombinant Receptor was produced as a fusion protein with affinity tags and it was expressed at a level of 3.5 pmol/mg of total membrane protein. [ 3 H]Bradykinin binding analysis revealed that the Recombinant Receptor binds to its endogenous ligand bradykinin with high affinity ( K d = 0.87 ± 0.1 nM), similar to the native Receptor. Enzymatic deglycosylation revealed that the Recombinant B 2 R was produced in a glycosylated form. Immunogold staining of the Pichia cells expressing B 2 R suggested that the Recombinant Receptor was localized intracellularly and it was not present in the plasma membrane. The data presented here should facilitate isolation of the Recombinant Receptor for structural studies.
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heterologous expression and comparative characterization of the human neuromedin u subtype ii Receptor using the methylotrophic yeast pichia pastoris and mammalian cells
The International Journal of Biochemistry & Cell Biology, 2007Co-Authors: Arun K. Shukla, Christoph Reinhart, Winfried Haase, Hartmut MichelAbstract:Abstract Neuromedin U (a neuropeptide) plays regulatory roles in feeding, anxiety, smooth muscle contraction, blood flow and pain. The physiological actions of NmU are mediated via two recently identified G protein-coupled Receptors namely the neuromedin U type 1 Receptor (NmU1R) and the neuromedin U type 2 Receptor (NmU2R). Despite their crucial roles in cell physiology, structural information on these Receptors is limited, mainly due to their low expression levels in native tissues. Here, we report the overexpression of the human NmU2R in the methylotrophic yeast Pichia pastoris and baby hamster kidney (BHK) cells using the Semliki Forest virus (SFV) system. The Recombinant Receptor was expressed as a fusion protein with three different affinity tags namely, the Flag tag, the histidine 10 tag and the biotinylation domain of Propionobacterium shermanii. Expression level of the Recombinant Receptor was 6–9 pmol/mg under optimized conditions, which is significantly higher than the expression level in the native tissues. The Recombinant Receptor binds to its endogenous ligand neuromedin U with high affinity (Kd = 0.8–1.0 nM) and the binding constant for the Recombinant Receptor is similar to that of the wild type NmU2R. Enzymatic deglycosylation suggested that the Recombinant NmU2R was glycosylated in P. pastoris, but not in BHK cells. Confocal laser scanning microscopy and immunogold labelling experiment revealed that the Recombinant Receptor was predominantly localized in the intracellular membranes. To our knowledge, this is the first report of heterologous overexpression of an affinity tagged Recombinant NmU2R and it should facilitate further characterization of this Receptor.
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Functional overexpression and characterization of human bradykinin subtype 2 Receptor in insect cells using the baculovirus system.
Journal of cellular biochemistry, 2006Co-Authors: Arun K. Shukla, Christoph Reinhart, Winfried Haase, Hartmut MichelAbstract:Bradykinin exerts its actions via binding to B1 and B2 Receptors (B1R and B2R), which are members of G protein-coupled Receptor superfamily. B2R is constitutively expressed in a variety of cells such as endothelial cells, vascular smooth muscle cells, and cardiomyocytes and it is an important drug target for the treatment of cardiovascular disorders. During this study, the human B2R was functionally overexpressed in insect cells using the baculovirus expression system. The maximum expression level in Sf9 cells under optimized condition was 10 pmol/mg. This corresponds to approximately 0.25 mg active Receptor per liter culture. The Recombinant Receptor showed high affinity for its endogenous ligand bradykinin, similar to the B2R expressed in native tissues. Functional coupling of the Recombinant Receptor to the endogenous Gαs protein was demonstrated via cAMP release assay upon agonist stimulation. Confocal laser scanning microscopy and immunogold-labeling experiment revealed that the Recombinant B2R was mainly localized intracellularly and only a minor fraction of the Recombinant Receptor reached the plasma membrane. To our knowledge, this is the first report of high level expression of Recombinant B2R in insect cells and provides a way for large scale production and structural characterization of this Receptor. J. Cell. Biochem. 99: 868–877, 2006. © 2006 Wiley-Liss, Inc.
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comparative analysis of the human angiotensin ii type 1a Receptor heterologously produced in insect cells and mammalian cells
Biochemical and Biophysical Research Communications, 2006Co-Authors: Arun K. Shukla, Christoph Reinhart, Hartmut MichelAbstract:Abstract Angiotensin II type 1a Receptor (AT 1a R) is a member of GPCR superfamily and it plays crucial roles in the regulation of blood pressure, hormone secretion and renal functions. Here, we report functional overexpression and characterization of the human AT 1a R in insect cells using the baculovirus system and in mammalian cells using the Semliki Forest virus system. The Recombinant Receptor was expressed at a level of 29–32 pmol/mg and it binds to angiontensin II with high affinity ( K d = 0.98–1.1 nM). Angiotensin II stimulated accumulation of inositol phosphate and phosphorylation of MAP kinase was also observed, which indicated that the Recombinant AT 1a R could couple to endogenous Gα q protein. Confocal laser scanning microscopy revealed that the Recombinant Receptor was predominantly localized in the plasma membrane and agonist induced internalization of the Recombinant AT 1a R was also observed. The Recombinant AT 1a R was expressed in glycosylated form and in vivo inhibition of glycosylation suppressed its surface expression.
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biochemical and pharmacological characterization of the human bradykinin subtype 2 Receptor produced in mammalian cells using the semliki forest virus system
Biological Chemistry, 2006Co-Authors: Arun K. Shukla, Christoph Reinhart, Winfried Haase, Hartmut MichelAbstract:Bradykinin, a vasoactive peptide, plays a crucial role in many cardiovascular processes via activation of the bradykinin subtype 2 Receptor (B2R). B2R, a member of the G protein-coupled Receptor (GPCR) superfamily, is a potential drug target in the treatment of cardiovascular disorders, pain and inflammation. In this study, human B2R was expressed at high levels in baby hamster kidney (BHK) cells using Semliki Forest virus-based vectors. The Recombinant Receptor was produced as a fusion protein with affinity tags and an expression level of 11 pmol/mg (i.e., approx. 0.2 mg of active Receptor per liter of culture) was obtained. Radioligand binding analysis revealed that the Recombinant Receptor binds to its endogenous ligand bradykinin with high affinity (Kd = 0.12 nM) and its pharmacological profile was similar to that of B2R in native tissues. Bradykinin-stimulated accumulation of inositol phosphate was observed in BHK cells expressing the Recombinant Receptor, which indicated the activation of endogenous G alpha(q) protein by the Recombinant B2R. Confocal laser scanning microscopy and immunogold staining revealed that the Recombinant Receptor was predominantly localized intracellularly. To the best of our knowledge, this is the first report of an affinity-tagged Recombinant B2R been expressed at high levels in BHK cells and extensively characterized.
Jeroen Pollet - One of the best experts on this subject based on the ideXlab platform.
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yeast expressed sars cov Recombinant Receptor binding domain rbd219 n1 formulated with aluminum hydroxide induces protective immunity and reduces immune enhancement
Vaccine, 2020Co-Authors: Wenhsiang Chen, Xinrong Tao, Anurodh S Agrawal, Abdullah Algaissi, Bihung Peng, Jeroen Pollet, Ulrich StrychAbstract:We developed a severe acute respiratory syndrome (SARS) subunit Recombinant protein vaccine candidate based on a high-yielding, yeast-engineered, Receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high levels of neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ~30% mortality in mice immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation with Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.
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yeast expressed sars cov Recombinant Receptor binding domain rbd219 n1 formulated with alum induces protective immunity and reduces immune enhancement
bioRxiv, 2020Co-Authors: Wenhsiang Chen, Maria Elena Bottazzi, Peter J. Hotez, Xinrong Tao, Anurodh S Agrawal, Abdullah Algaissi, Bihung Peng, Jeroen Pollet, Ulrich Strych, Sara LustigmanAbstract:We developed a severe acute respiratory syndrome (SARS) subunit Recombinant protein vaccine candidate based on a highyielding, yeast- engineered, Receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ~ 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and other emerging and re-emerging beta-CoVs such as SARS-CoV-2.
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optimization of the production process and characterization of the yeast expressed sars cov Recombinant Receptor binding domain rbd219 n1 a sars vaccine candidate
Journal of Pharmaceutical Sciences, 2017Co-Authors: Wenhsiang Chen, Jeroen Pollet, Shivali M Chag, Mohan Vivekanandan Poongavanam, Amadeo B Biter, Ebe A Ewere, Wanderson Rezende, Christopher A Seid, Elissa M HudspethAbstract:From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to 5 continents and caused 8000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a Recombinant protein vaccine candidate is under development. Previously, we reported the 5 L scale expression and purification of a promising Recombinant SARS vaccine candidate, RBD219-N1, the 218–amino acid residue Receptor-binding domain (RBD) of SARS coronavirus expressed in yeast–Pichia pastoris X-33. When adjuvanted with aluminum hydroxide, this protein elicited high neutralizing antibody titers and high RBD-specific antibody titers. However, the yield of RBD219-N1 (60 mg RBD219-N1 per liter of fermentation supernatant; 60 mg/L FS) still required improvement to reach our target of >100 mg/L FS. In this study, we optimized the 10 L scale production process and increased the fermentation yield 6- to 7-fold to 400 mg/L FS with purification recovery >50%. A panel of characterization tests indicated that the process is reproducible and that the purified, tag-free RBD219-N1 protein has high purity and a well-defined structure and is therefore a suitable candidate for production under current Good Manufacturing Practice and future phase-1 clinical trials.
Wenhsiang Chen - One of the best experts on this subject based on the ideXlab platform.
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yeast expressed sars cov Recombinant Receptor binding domain rbd219 n1 formulated with aluminum hydroxide induces protective immunity and reduces immune enhancement
Vaccine, 2020Co-Authors: Wenhsiang Chen, Xinrong Tao, Anurodh S Agrawal, Abdullah Algaissi, Bihung Peng, Jeroen Pollet, Ulrich StrychAbstract:We developed a severe acute respiratory syndrome (SARS) subunit Recombinant protein vaccine candidate based on a high-yielding, yeast-engineered, Receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high levels of neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ~30% mortality in mice immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation with Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.
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yeast expressed sars cov Recombinant Receptor binding domain rbd219 n1 formulated with alum induces protective immunity and reduces immune enhancement
bioRxiv, 2020Co-Authors: Wenhsiang Chen, Maria Elena Bottazzi, Peter J. Hotez, Xinrong Tao, Anurodh S Agrawal, Abdullah Algaissi, Bihung Peng, Jeroen Pollet, Ulrich Strych, Sara LustigmanAbstract:We developed a severe acute respiratory syndrome (SARS) subunit Recombinant protein vaccine candidate based on a highyielding, yeast- engineered, Receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ~ 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and other emerging and re-emerging beta-CoVs such as SARS-CoV-2.
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optimization of the production process and characterization of the yeast expressed sars cov Recombinant Receptor binding domain rbd219 n1 a sars vaccine candidate
Journal of Pharmaceutical Sciences, 2017Co-Authors: Wenhsiang Chen, Jeroen Pollet, Shivali M Chag, Mohan Vivekanandan Poongavanam, Amadeo B Biter, Ebe A Ewere, Wanderson Rezende, Christopher A Seid, Elissa M HudspethAbstract:From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to 5 continents and caused 8000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a Recombinant protein vaccine candidate is under development. Previously, we reported the 5 L scale expression and purification of a promising Recombinant SARS vaccine candidate, RBD219-N1, the 218–amino acid residue Receptor-binding domain (RBD) of SARS coronavirus expressed in yeast–Pichia pastoris X-33. When adjuvanted with aluminum hydroxide, this protein elicited high neutralizing antibody titers and high RBD-specific antibody titers. However, the yield of RBD219-N1 (60 mg RBD219-N1 per liter of fermentation supernatant; 60 mg/L FS) still required improvement to reach our target of >100 mg/L FS. In this study, we optimized the 10 L scale production process and increased the fermentation yield 6- to 7-fold to 400 mg/L FS with purification recovery >50%. A panel of characterization tests indicated that the process is reproducible and that the purified, tag-free RBD219-N1 protein has high purity and a well-defined structure and is therefore a suitable candidate for production under current Good Manufacturing Practice and future phase-1 clinical trials.