The Protein Expression Unit at the Structure Proteomics Unit (SPU) expresses and produces recombinant proteins from diverse biological sources. Genes of interest, cloned into suitable expression vectors, are introduced into host cells to produce recombinant proteins. To increase the probability that a given protein will be expressed in a soluble and active form, and in high yield, several expression systems are used. These include bacterial (primarily E. coli) and two eukaryotic systems - baculovirus-infected insect cells (Sf9 and Tni/High5) and mamalian Expi293/HEK293T cells.
Unit activities
Small scale expression tests:
Expression screening in E. coli.
Baculovirus production and small scale expression screening in insect cells (Sf9 or Tni).
Expression screening in mamalian cells (HEK293).
Complex expression and protein interaction
- · Multi-complex expression in insect cells using the MultiBac system
· Protein interaction analysi9s by pull-down assays
Production of in-vivo biotinylated proteins
Large scale production under optimized conditions in the appropriate expressoion system.
E. coli large scale expression (5L)
Insect cells expression (1.5L)
Mammalian cell expression (0.3L)
Partial purification Analysis of soluble and insoluble fractions using:
Protein purification of proteins from small scale expression test using the following affinity tags- His-tag, Flag-tag, Strep-tag, HA-tag, Monomeric Avidin, etc…
SDS-PAGE
Western Blot with specific antibodies
Consultation
- Selection of optimal expression systems and host cell-lines for target proteins
- Guidence on affinity tag selecxtion and positioning.
- Expression strategies for full-length proteins and /or specific domains
- Codon-optimization
- Troubleshooting for low or failed expression.