Advanced methods for recombinant production of challenging proteins
#wemakeproteins
Description
The increasing complexity of biological targets subject to structural characterization constantly demands highly versatile approaches of recombinantly expression and purification. When targeting macromolecular complexes, the difficulties associated to the initial recombinant screening phase are further amplified by the need to identify suitable constructs to co- express or reconstitute in vitro the molecular interactions essential for complex stabilization. Large industry-scale high-throughput platforms offer extremely efficient and automated methods to obtain libraries of protein constructs for expression and purification scouting, whereas several academic research labs still rely on more conservative “one construct, one recombinant host, one target” approaches. Frequently, the choice depends on investments in automation, as the costs associated to creation of high-throughput facilities are not affordable to all research groups. Aiming at minimizing time and costs associated to the initial screening for recombinant expression, we optimized cloning and expression strategies to increase the throughput without the need for automation, and we coupled them to an advanced facility which includes bacterial, yeast, insect, and mammalian cell expression hosts.
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HEK293 cells grown in suspension in our mammalian cell protein expression laboratory, part of the recombinant protein facility we established. |
Publications and Dissemination
Publications
- Faravelli S*, Campioni M, Palamini M, Canciani A, Chiapparino A, Forneris F.* Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension. Bio-Protocol, 11, e3998. (2021) *Shared Corresponding Authors – PubMed – UNIFIND
- Forneris F, Canciani A, Ciossani G, Palamini M, Chiapparino A, Gabrieli P, Guarino SR, Campioni M, Nenci S, Magnani F. Versatile medium-throughput strategies for recombinant expression screening in structural biology. Acta Crystallographica A, A73, C1276. (2017) – (IUCR 2017 conference abstract)