Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension

Abstract

Recombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In this regard, human embryonic kidney 293 (HEK293) cells constitute one of the main standard lab-scale mammalian hosts for recombinant protein production since these cells are relatively easy to handle, scale-up, and transfect. Here, we present a detailed protocol for the cost-effective, reproducible, and scalable implementation of HEK293 cell cultures in suspension (suitable for commercially available HEK293 cells, HEK293-F) for high-quantity recombinant production of secreted soluble multi-domain proteins. In addition, the protocol is optimized for a Monday-to-Friday maintenance schedule, thus simplifying and streamlining the work of operators responsible for cell culture maintenance.

Epidemic Preparedness—Leishmania tarentolae as an Easy-to-Handle Tool to Produce Antigens for Viral Diagnosis: Application to COVID-19

“…Finally, our study underlines L. tarentolae as a system that is easy to manipulate and culture, providing a sound alternative for the rapid production of serodiagnostic proteins, even at the forefront of novel epidemics, e.g., in the presence of “spillover” events in tropical countries ( Olival et al, 2017 ). For example, the production of proteins by Leishmania cells can be obtained in flasks or simple bioreactors, within 2–4 days from the starting of the culture ( Fritsche et al, 2007 ), whereas mammalian systems, such as the HEK cells, require longer times 4–6 days ( Faravelli et al, 2021 ). We emphasize that protein production in L. tarentolae holds the potential to be scaled to the industrial level, growing the parasites in bioreactors and harvesting proteins using high throughput strategies ( Niimi, 2012 ).…”

“…The pCAGGS plasmid for production of the C-terminal His-tagged SARS-CoV-2 Spike RBD (#NR_52310) was obtained from BEI Resources (NY, United States). Recombinant SARS-CoV-2 Spike RBD was produced using HEK293-F cells (Invitrogen) cultivated in suspension using FreeStyle medium (Invitrogen) as described in Faravelli et al (2021) . The cell medium containing secreted SARS-Cov2 Spike RBD was collected 6 days after transfection.…”

SARS-CoV-2 Spike Affinity and Dynamics Exclude the Strict Requirement of an Intermediate Host

“…Recombinant hACE2 ectodomain, affi ACE2 ectodomain, SARS-CoV-2 RBD, and RaTG13 RBD were produced using HEK293-F cells (Invitrogen) cultivated in suspension using Freestyle medium (Invitrogen) as described in ( 35 ). Briefly, cells were transfected at a cell density of 1 million mL −1 using a mixture of 1 μg of recombinant expression plasmid and 3 μg of polyethyleneimine (PEI; Polysciences, Germany).…”

“…Freestyle medium (Invitrogen) as described in (35). Briefly, cells were transfected at a cell density of 1 million mL −1 using a mixture of 1 μg of recombinant expression plasmid and 3 μg of polyethyleneimine (PEI; Polysciences, Germany).…”

A Pilot Study on Covid and Autism: Prevalence, Clinical Presentation and Vaccine Side Effects

“…To detect specific antibodies in university laboratories, recombinant SARS-CoV-2 spike receptor binding domain (RBD) was produced using polyethyleneimine-based transient transfection of Freestyle HEK293 Cells (Life Technologies) cultivated in suspension according to Faravelli and colleagues [13]. The pCAGGS plasmids for production of the C-terminal His-tagged SARS-CoV-2 Spike RBD (#NR_52310) were obtained from BEI Resources (NY, USA).…”