Bacteria
As a service-dedicated national infrastructure, PPS offers protein production services in various Escherichia coli hosts.
With two specialized facilities (PSF and PEP, Figure 1.), PPS offers much needed services and expert advice at all stages involved in protein production in E. coli. PPS keeps stocks of more than 20 different ready-to-use cloning vectors (containing a variety of fusion proteins and purification tags) designed to improve protein expression levels and to facilitate protein purification. E. coli expression cultures, usually 1-8 liters depending on the expected yield, are cultivated. Cultures are grown in TB or LB at 37°C before down-tempering to 18-20 °C. After induction protein production is allowed to continue for a couple of hours or overnight. What PPS offer:
A. Different expressions strains
BL21(DE3), BL21plysS (DE3), Rosetta (DE3), Origami2(DE3), Arctic Express(DE3), C41(DE3), C43(DE3), XL Gold, BL21, BL21 Star (DE3), Shuffle T7 express and BL21-AI.
B. Small scale expression and solubility test
Small scale expression test where the levels of total protein as well as the yield after affinity purification are assessed by SDS-PAGE analysis. Typically, 1 ml cultures are grown in 96 well blocks in Terrific Broth with expression at 18°C and overnight. Harvested cells are lysed and clarified supernatants are subjected to an IMAC affinity step. Work with several different E. coli strains under different expression conditions facilitates an optimal output.
C. Large scale expression
1-16 liter culture in LB or TB media (larger volumes are up for discussion)
D. Protein Labelling
Labelling such as 15N, 13C, SeMet, biotinylation and unnatural amino acids (UAA).
As well as subsequent protein purification and characterizations
Figure 1.
Snapshots of PPS facilities
