Expression and processing of hepatitis C virus structural proteins in Pichia pastoris yeast

Martínez‐Donato, Gillian; Acosta‐Rivero, Nelson; Morales-Grillo, Juan; Musacchio, Andrea; Viña, Ariel; Álvarez, Catalina; Figueroa, Nelvis; Guerra, Ignacio Ruiz; García, José; Varas, Laura; Muzio, Verena; Dueñas‐Carrera, Santiago

doi:10.1016/j.bbrc.2006.01.157

Cited by 17 publications

(17 citation statements)

References 27 publications

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“…False positive deamidation commonly occurs in small and hydrophilic amino acids such as glycine and serine at position X of the NX(S/T/C) consensus sequence (16). Although LILRA3 does not contain such consensus sequences, we performed stringent control experiments using E. coli-produced rLILRA3 with or without PNGase treatment and PNGase-untreated mammalian rLILRA3-His.…”

Section: Discussionmentioning

confidence: 99%

“…Glycosylation-PNGase F cleaves between N-acetylglucosamine (GlcNAc) and asparagine (Asn) residues on N-linked high mannose glycans, hybrid glycans, and complex glycans (16). Treatment of recombinant LILRA3 produced in 293T cells and P. pastoris with this enzyme reduced their molecular mass to a size similar to the non-glycosylated rLILRA3 produced in E. coli ( Fig.…”

Section: The Increase In the Molecular Mass Of Lilra3 Produced In Mammentioning

confidence: 99%

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Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

Lee

Mitchell

Lau

et al. 2013

Journal of Biological Chemistry

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Background: LILRA3 is a soluble receptor with unknown functions that is abundantly present in human serum. Results: Optimally glycosylated recombinant LILRA3 protein produced only in a mammalian system binds potential ligands and suppresses monocyte function. Conclusion: LILRA3 suppresses LPS-mediated TNF production, suggesting that it is a new anti-inflammatory protein.Significance: This work provides first insight into the biochemical characteristics and functions of LILRA3.

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Section: Discussionmentioning

confidence: 99%

Section: The Increase In the Molecular Mass Of Lilra3 Produced In Mammentioning

confidence: 99%

Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

Lee

Mitchell

Lau

et al. 2013

Journal of Biological Chemistry

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“…Initially, COOHterminal truncated versions of E2, based on those described previously (41), were displayed on the surface of yeast, and MAbs were tested for immunoreactivity by flow cytometry (Fig. 6) (see Table S1 in the supplemental material).…”

Section: Epitope Localization Of Mabsmentioning

confidence: 99%

Neutralizing Monoclonal Antibodies against Hepatitis C Virus E2 Protein Bind Discontinuous Epitopes and Inhibit Infection at a Postattachment Step

Sabo

Luca

Prentoe

et al. 2011

J Virol

116

135

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The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To characterize the structural and functional basis of HCV neutralization, we generated a novel panel of 78 monoclonal antibodies (MAbs) against E2 proteins from genotype 1a and 2a HCV strains. Using high-throughput focus-forming reduction or luciferase-based neutralization assays with chimeric infectious HCV containing structural proteins from both genotypes, we defined eight MAbs that significantly inhibited infection of the homologous HCV strain in cell culture. Two of these bound E2 proteins from strains representative of HCV genotypes 1 to 6, and one of these MAbs, H77. Hepatitis C virus (HCV) is a blood-borne hepatotropic virus that infects ϳ170 million people worldwide. Approximately 70% of infected individuals progress to chronic liver disease, which carries an increased risk of cirrhosis and hepatocellular carcinoma (7). In general, treatment of chronic HCV infection is complicated by resistance due to extensive genetic diversity. HCV has been classified into seven major genotypes, which differ by ϳ30% at the nucleotide level (4), and this positivesense, single-stranded RNA virus has a capacity for rapid evolution of variant viruses during persistent infection. The current treatment, pegylated ␣ 2a interferon (IFN-␣ 2a ) and ribavirin, has variable side effects and response rates depending on the virus and host genotype (16). No vaccine is currently available, and preclinical development has been hampered by a lack of understanding of which conserved epitopes on the HCV structural proteins should be targeted.HCV contains an ϳ9.6-kb RNA genome that is translated as a single polyprotein and then cleaved by viral and host proteases into structural proteins (core, E1, and E2), p7, and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (39). Viral attachment and entry are mediated by the envelope glycoproteins, E1 and E2. Four attachment or entry receptors that are required for infection of hepatocytes have been identified, including CD81 (53), scavenger receptor B1 (SR-B1) (56), and the tight-junction proteins claudin 1 (CLDN1) (14) and occludin (OCLN) (54). The importance of E2 binding to the large extracellular loop of CD81 has been established in vitro (13,18,28,50,53), and interactions between E2 hypervariable region 1 (HVR1) and SR-B1

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“…Indeed, the conservative feature of the core gene in different HCV genotypes and the low susceptible of mutation in this region, make this protein an ideal candidate for HCV vaccine (2). Therefore, the production of core protein was done in different expression systems (12)(13)(14)(15)(16)(17). The use of bacterial systems showed promising results as compared to other eukaryotic systems such as insect cells and yeast because of less time for production, high-level yield, and cost-effectiveness ( (3)).…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, the use of recombinant proteins especially structural proteins can be considered as an important strategy in stimulation of humoral immune responses. Many studies have produced HCV core or HCV core-E1E2 proteins in different expression systems such as bacteria (12,13), insects (4,10,14), and yeast (15,16). Usually, the truncated fragments of core, E1 or E2 were produced in bacterial systems (17).…”

mentioning

confidence: 99%

Expression and Purification of HCV Core and Core-E1E2 Proteins in Different Bacterial Strains

et al. 2015

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Expression and processing of hepatitis C virus structural proteins in Pichia pastoris yeast

Cited by 17 publications

References 27 publications

Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

Neutralizing Monoclonal Antibodies against Hepatitis C Virus E2 Protein Bind Discontinuous Epitopes and Inhibit Infection at a Postattachment Step

Expression and Purification of HCV Core and Core-E1E2 Proteins in Different Bacterial Strains

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