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.

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. We are 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.  Questions, input, and suggestions are welcome.

The protein production platform is a national facility whitin SciLifeLab and SWEDSTRUCT.


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 (ref1ref2). 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
(e.g. Rosetta2).

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.

Cloning vectors

Expression plasmids for E. coli

pNIC-NHCS, Strep II Tag

pNIC-Bsa4,  cleavable N-terminal His-tag

pNIC-CH2, C-terminal His-tag

pNIC-Trx, separately cleavable Thioredoxin fusion in front of cleavable His-tag

pNIC-GST, separately cleavable GST fusion in front of cleavable His-tag

pNIC-MBP3, separately cleavable MBP fusion in front of cleavable His-tag

pNIC-BASY, N-terminal OsmY for secretion to media in front of cleavable His-tag

pNIC28-DsbA, N-terminal DsbA1 for direction to periplasm, in front of cleavable His tag

pNIC28-PelB, N-terminal PelB for direction to periplasm, in front of cleavable His tag

pNIC-Bio3, cleavable N-terminal His-tag, C-terminal biotinylation site

pACYC-Bsa1, cleavable N-terminal His-tag, CamR

pACYC-FLAG, cleavable N-terminal  FLAG-tag, CamR

pNIC-eCFP, cleavable N-terminal His-tag, C-terminal eCFP

pNIC-eYFP, cleavable N-terminal His-tag, C-terminal eYFP

pNIC-eGFP, cleavable N-terminal His-tag, C-terminal eGFP




Expression plasmids for other hosts

pcDNA5-CF-LIC, regulated by tetracycline,  C-terminal FLAG-tag

pcDNA3-CTHF-LIC, cleavable C-terminal His-FLAG tag

pcDNA3-N-Flag-LIC, N-terminal FLAG tag

pcDNA3-C-GFP-LIC, C-terminal GFP

pFB-LIC-Bse (Bac-to-Bac compatible), cleavable N-terminal His- tag

pVL1392-LIC-Bse (BaculoGold/FlashBAC compatible), cleavable N-terminal His- tag

pVL1392-CH2 (BaculoGold/FlashBAC compatible), C-terminal His-tag


..more vectors coming

Related protein production platforms

Umeå University

Protein Expertise Platform

Göteborg University

Mammalian Protein Expression

Cell-free Protein Expression

Lund University

Lund Protein-Production Platform

Royal Institute of Technology

Bioprocess plant facility


Protein Expression and Characterization Facility



Senior Lecturer

Helena Berglund

Phone: +46-(0)8-524 868 43
Organizational unit: Protein Science Facility