Protein Production Platform
The PSF set-up is built on the protein production platform of the Stockholm node of the Structural Genomics Consortium and focus on molecular biology and protein purification and specifically production of His tagged proteins in E. coli.
Our plasmid collection contains vectors for protein production with different fusion proteins in different hosts. E. coli cultures (including Se-Met labeling, isotope labeling for NMR, biotinylation, etc) is performed in-house while we collaborate with other expert platforms if other expression hosts are required.
We have a high throughput pipeline but will also take on more tailor made projects when time permits. We are also happy to perform purifications of a large set of proteins structure determined within the Structural Genomics Consortium, as well as utility proteins such as e.g. recombinant TEV-protease and Tn5.
All questions, input, and suggestions are welcome!
Cloning and small scale expression tests
A multi-construct approach is applied to the construct design: Small variations in the start and stop positions of a protein can change the biophysical properties of the expressed protein as well as expression efficiency, purification yield, and crystallisation properties.
Therefore we prefer to clone and expression screen several variants of a targeted domain in parallel (ref1, ref2). Usually 3 to 4 start and stop positions are designed for each targeted domain based on previous knowledge or predictions of the protein structure. We are happy to help with the construct design.
High throughput cloning
In ligation independent cloning, complementary ssDNA overhangs on plasmid and PCR products are generated by T4 DNA polymerase. After annealing and transformation into E. coli the presence of a correctly sized insert is verified by colony PCR and the insert is sequenced after a plasmid preparation step. The method is robust and enables parallel cloning of many constructs in a 96 well format. On average the cloning success rate is 87%.
Our most frequently used cloning vector is pNIC28-Bsa4 which adds an N-terminal His-tag followed by a TEV protease cleavage site to the target protein. For expression we use BL21(DE3) E. coli strains, also strains that compensate for different codon usages
Small scale expression screening
All positive clones are subjected to a 1 ml small scale expression test where the levels of total protein as well as the yield after affinity purification are assessed by SDS-PAGE analysis. The cultures are grown in 96 well blocks in Terrific Broth at 37°C before down tempering to 18°C and induction of expression over night. Harvested cells are lysed and subjected to an IMAC affinity step, also this in 96 well format.
Estimated time for cloning and small scale expression screening is one month, starting when the lab-work is initiated.
Large scale cultures
Depending on the small scale expression results or previously known expression efficiency, 0.75 – 4.5 liters of culture is grown in TB in a LEX system. Cultures are typically grown at 37°C before down-tempering to 18 °C and induction of protein production with IPTG over night.
High throughput protein purification
Harvested cells are stored at -80°C before lysis by sonication, clarification by centrifugation and filtering prior to applying the sample to an ÄKTAxpress system for a two step purification. Our standard purification scheme consists of an IMAC affinity step utilizing the His-tag followed by a size exclusion chromatography step (SEC, gel filtration). The final step is generally performed in a buffer consisting of 20 mM HEPES, 300 mM NaCl, 10% glycerol and 0.5 mM TCEP at a pH of 7.5, this buffer that has agreed well with a majority of the SGC Stockholm proteins and that has become our first choice.
The purity if of the purified protein is assessed by SDS- PAGE, the mass with mass spectrometry (at KTH), and an overall judgment of the protein behavior is also achieved from the appearance of the gel filtration elution profile.
Polishing purification steps
Based on the purification results we can suggest, and after agreement perform, additional steps to improve the yield or purity.