SARS-CoV-2 Spike (S) glycoprotein expression, purification and characterization in suspension human embryonic kidney cells 293
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2021
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PROTEIN EXPRESSION IN ANIMAL CELLS CONFERENCE, 15th
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SARS-CoV-2 is a zoonotic virus RNA positive, which became responsible to be the
largest sanitary crisis faced by humanity: Coronavirus disease 2019 (COVID-19). Some
symptoms include major sneeze conditions, who could evolve to severe acute respiratory
syndrome, and in some cases, to death. Techniques for accurate detection of this virus are
essential to promote an accurate diagnosis of infected patients. SARS-CoV-2 has several
targets with clinical interest; although, the focuses is on Spike (S), a homotrimer
glycoprotein, that interacts with angiotensin converting enzyme receptor (ACE2),
developing the infection in host cells. Thus, we recognize that the demand for the
glycoprotein S is necessary, requiring large amounts with high purity level. The current
work has the main objective the transient expression of SARS-CoV-2 S protein into
suspension human embryonic kidney cells 293 (HEK293), purification and
characterization, to use it as a template for discovering new molecular markers. SARSCoV-
2 S modified protein cDNA was inserted into pαH plasmid, amplified, and purified.
For transient recombinant protein expression, 7.5 x 107
HEK293 cells (Expi293FTM cells)
was seeded in 27 mL Expi293™ culture Medium. The transfection was carried out with a
cationic lipid ExpiFectamine™ and 30 μg of plasmid, mixed with 3 mL Opti-Mem®
culture medium. Cell culture was maintained for seven days in 125 mL vent cap
Erlenmeyer, 32 ºC, 8% CO2, under 125 rpm orbital shaker rotation. 10 mL aliquots were
collected on four- and seven-day post transfection, stored at -80 ºC. Physical-chemical
and biological characterizations were determined by SDS-PAGE, ELISA, and Western
Blotting. Purification from 40 mL of conditioned medium was carried out in two steps:
Strep-Tag affinity column, followed by a size exclusion Superose® 6 (10/300), 5.0 mg of
oligomeric recombinant protein with 95% purity was obtained. We believe that this
process can be easily adapted to different volumes, being very useful for obtaining, in a
short time, enough pure and immunological active SARS-CoV-2 S for further studies and
applications, such as, cryogenic electron microscopy, mass spectroscopy, N-glycan
structures, antibody production and immunologic assays development.
Como referenciar
FREIRE, RENAN P.; SILVA, FELIPE D.; VITALE, PHELIPE; TODESCHINI, IRIS; MENEZES, FILIPE; OLIVEIRA, JOAO E. de; OLIVEIRA, MONA das N.; SOARES, CARLOS R.J. SARS-CoV-2 Spike (S) glycoprotein expression, purification and characterization in suspension human embryonic kidney cells 293. In: PROTEIN EXPRESSION IN ANIMAL CELLS CONFERENCE, 15th, September 13-16, 2021, Online. Abstract... Disponível em: http://repositorio.ipen.br/handle/123456789/32906. Acesso em: 25 Apr 2024.
Esta referência é gerada automaticamente de acordo com as normas do estilo IPEN/SP (ABNT NBR 6023) e recomenda-se uma verificação final e ajustes caso necessário.