In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI–MS spectrum

Espinosa, Luis Ariel; Ramos, Yassel; Andújar, Iván; Torres, Enso Onill; Cabrera, Gleysin; Martín, Alejandro; Roche, D.G.; Chinea, Glay; M, Becquet; González, Isabel; Canaán-Haden, Camila; Nelson, Elías; Rojas, Gertrudis; Pérez-Massón, Beatriz; Pérez-Martínez, Dayana; Boggiano, Tammy; Palacio, Julio; Chang, Sum Lai Lozada; Hernández, Lourdes; Hernández, Kathya Rashida de la Luz; Saloheimo, Markku; Vitikainen, Marika; Valdés-Balbín, Yury; Santana-Medero, Darielys; Rivera, Daniel G.; Vérez-Bencomo, Vicente; Emalfarb, Mark; Tchelet, Ronen; Guillén, Gerardo; Limonta, Miladys; Pimentel, Eulogio; Ayala, Marta; Besada, Vladimir; González, Luís Javier Durán

doi:10.1007/s00216-021-03721-w

Cited by 12 publications

(23 citation statements)

References 63 publications

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“…Authors concluded that the protein has three predominant glycoforms, Man4, Man4A1 and Man5A1, the last one being predominant. In addition, the electrospray ionization mass spectrometry permitted full-sequence coverage and confirmed the integrity of the N- and C-terminal ends and the correct conformation of the four native disulfide bonds [22] .…”

Section: Discussionmentioning

confidence: 88%

“…Previously, the RBDf was characterized by mass spectrometry using an in-solution buffer-free digestion protocol [22] . Authors concluded that the protein has three predominant glycoforms, Man4, Man4A1 and Man5A1, the last one being predominant.…”

Section: Discussionmentioning

confidence: 99%

“…The construct was transformed into the fungus Thermothelomyces heterothallica (formerly Myceliophthora thermophila ), C1 strain DNL155, and it was integrated into the bgl8 locus. The RBDf protein has been expressed at greater than 2 g/l levels in 120 h in fed-batch fermentation and it was purified using a CaptureSelect™ C-tag affinity resin coupled to a camelid single-chain antibody, called NbSyn2, reaching a high level of purity [11] . The protein sequence has 204 aa and starts after the first N-glycosylation site (RBD amino acids 333–527 of the S2 protein).…”

Section: Methodsmentioning

confidence: 99%

“…To evaluate the immune response induced by the RBDf protein, BALB/c mice were immunized with different preparations by subcutaneous and intranasal administration routes ( Table 1 ). As a control, the RBD expressed in the HEK293 cell line was also evaluated [11] . The HBV core protein was co-administered with the RBD proteins, taking into account its immune-enhancement capacity previously described.…”

Section: Immunological Evaluation In Balb/c Micementioning

confidence: 99%

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A recombinant SARS-CoV-2 receptor-binding domain expressed in an engineered fungal strain of Thermothelomyces heterothallica induces a functional immune response in mice

Lazo

Bequet-Romero

Lemos

et al. 2022

Vaccine

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Since the beginning of the COVID-19 pandemic, the development of effective vaccines against this pathogen has been a priority for the scientific community. Several strategies have been developed including vaccines based on recombinant viral protein fragments. The receptor-binding domain (RBD) in the S1 subunit of S protein has been considered one of the main targets of neutralizing antibodies. In this study we assess the potential of a vaccine formulation based on the recombinant RBD domain of SARS-CoV-2 expressed in the thermophilic filamentous fungal strain Thermothelomyces heterothallica and the hepatitis B virus (HBV) core protein. Functional humoral and cellular immune responses were detected in mice. To our knowledge, this is the first report on the immune evaluation of a biomedical product obtained in the fungal strain T. heterothallica. These results together with the intrinsic advantages of this expression platform support its use for the development of biotechnology products for medical purpose.

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Section: Discussionmentioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Immunological Evaluation In Balb/c Micementioning

confidence: 99%

See 2 more Smart Citations

A recombinant SARS-CoV-2 receptor-binding domain expressed in an engineered fungal strain of Thermothelomyces heterothallica induces a functional immune response in mice

Lazo

Bequet-Romero

Lemos

et al. 2022

Vaccine

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“…The influenza HA shown here were not the only antigens that were successfully expressed in C1. Rift valley fever virus (RVFV) and Schmallenberg virus (SBV) viral antigens have been expressed at greater than 1.5 g/L in five days in the framework of the Zoonosis Anticipation Preparedness Initiative (ZAPI) [49] and C1 strains have been used to produce the SARS-CoV-2 receptor binding domain (RBD) at greater than 2 g/L in five days [50]. Additionally, C1 strains have also been rapidly developed that express the Alpha, Beta, Gamma, and Delta variant RBDs, the SARS-CoV-2 full spike protein, as well as an Fc-RBD.…”

Section: Discussionmentioning

confidence: 99%

The Highly Productive Thermothelomyces heterothallica C1 Expression System as a Host for Rapid Development of Influenza Vaccines

Keresztes¹,

Baer²,

Alfenito³

et al. 2022

Vaccines

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(1) Influenza viruses constantly change and evade prior immune responses, forcing seasonal re-vaccinations with updated vaccines. Current FDA-approved vaccine manufacturing technologies are too slow and/or expensive to quickly adapt to mid-season changes in the virus or to the emergence of pandemic strains. Therefore, cost-effective vaccine technologies that can quickly adapt to newly emerged strains are desirable. (2) The filamentous fungal host Thermothelomyces heterothallica C1 (C1, formerly Myceliophthora thermophila) offers a highly efficient and cost-effective alternative to reliably produce immunogens of vaccine quality at large scale. (3) We showed the utility of the C1 system expressing hemagglutinin (HA) and a HA fusion protein from different H1N1 influenza A virus strains. Mice vaccinated with the C1-derived HA proteins elicited anti-HA immune responses similar, or stronger than mice vaccinated with HA products derived from prototypical expression systems. A challenge study demonstrated that vaccinated mice were protected against the aggressive homologous viral challenge. (4) The C1 expression system is proposed as part of a set of protein expression systems for plug-and-play vaccine manufacturing platforms. Upon the emergence of pathogens of concern these platforms could serve as a quick solution for producing enough vaccines for immunizing the world population in a much shorter time and more affordably than is possible with current platforms.

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An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen

et al. 2022

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In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI–MS spectrum

Cited by 12 publications

References 63 publications

A recombinant SARS-CoV-2 receptor-binding domain expressed in an engineered fungal strain of Thermothelomyces heterothallica induces a functional immune response in mice

A recombinant SARS-CoV-2 receptor-binding domain expressed in an engineered fungal strain of Thermothelomyces heterothallica induces a functional immune response in mice

The Highly Productive Thermothelomyces heterothallica C1 Expression System as a Host for Rapid Development of Influenza Vaccines

An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen

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