PEPCF expresses proteins in bacteria, insect and mammalian cells and uses a variety of chromatographic and biophysical techniques for protein purification and characterization.
We currently work with 3 different host organisms for protein expression, which are E. coli, insect cells and mammalian cells.
We accept most standard expression vector backbones for protein expression in E. coli. For recombinant protein expression in insect cells, we work with baculovirus-mediated expression in lepidopteran insect cell lines (Sf9, Sf21, Hi5). For generating the recombinant baculoviruses, we make use of the Bac-to-Bac system, which means we can only accept constructs compatible with the Bac-to-Bac workflow. Regarding protein expression in mammalian cells, we offer transient transfection in HEK293F suspension cultures or baculovirus-mediated expression in mammalian cells using the BacMam system.
If you decide to express a recombinant protein in E. coli, the first step will be to design your expression construct and choose an expression vector. In E. coli, you can express your protein of interest in the cytoplasm or in the periplasm.
In E. coli, the cytoplasm is a reducing environment, whereas the periplasm is an oxidizing environment that allows the formation of disulfide bonds. The periplasm also contains the DsbA/DsbB oxido-reductases and the DsbC/DsbD disulfide bond isomerases. Therefore, the periplasm might be a more suitable compartment for the expression of disulfide bond-rich proteins in E. coli. Other advantages are the lower proteolytic activity in the periplasm and the presence of molecular chaperones such as FkpA, SurA and Skp. The periplasm only contains about 4% of the total cellular proteins and hence facilitates purification after an osmotic shock. The disadvantages are that protein expression in the periplasm often results in a lower yield than cytosolic expression and usually not all expressed protein will be secreted into the periplasm. To direct a recombinant protein to the periplasm, you need to add a periplasmic signal sequence to the N-terminus of your protein, which will be removed after crossing the inner membrane.
If you want to express multiple proteins simultaneously in E. coli, you can either go for monocistronic or polycistronic co-expression. Monocistronic means that each mRNA will encode for 1 protein, while in polycistronic co-expression 1 mRNA will encode for 2 or more proteins.
Co-expression can be useful if your protein of interest is part of a multi-subunit complex and might not be soluble on its own. Sometimes co-expression with chaperones or foldases can also help with in vivo protein folding. Some well-characterized chaperones are for example GroEL-GroES, DnaK-DnaJ-GrpE and tig. Chaperone plasmid sets for co-expression are also commercially available. Foldases that play an important role in protein folding are peptidyl prolyl cis/trans isomerases (PPI’s), disulfide oxidoreductase (DsbA), disulfide isomerase (DsbC) and protein disulfide isomerase (PDI).
When you co-express various proteins using multiple plasmids, the plasmids should have different antibiotic resistances and compatible origins of replication. Commercial systems for protein co-expression in E. coli are for example the pET-Duet suite and the MultiColi system.
In the pET-Duet suite, each vector has 2 individual gene expression cassettes, which allows you to co-express up to 8 proteins when combining vectors.
|Vector||Ori||Antibiotic resistance||Copy number|
A big advantage of protein expression in E. coli is the availability of a large variety of E. coli expression strains, that all have their own specific characteristics. Some strains possess extra copies of rare tRNA’s, which is very useful if the codon usage of your gene of interest is very different from the E. coli codon usage. Other strains are better equipped to deal with the expression of toxic proteins or are more suitable for the expression of disulfide bond-rich proteins in the cytoplasm. Usually, you screen a number of different expression strains and expression conditions in small scale expression and purification tests. In these small scale tests you’ll assess the total expression level of your protein of interest, the solubility and the ability to enrich it on affinity beads. Once you identify the best conditions, you can scale up your expression cultures and proceed with the large scale protein purification.
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Rosano G.L., Morales E.S. and Ceccarelli E.A. (2019) New tools for recombinant protein production in Escherichia coli: a 5-year update. Protein Science 28: 1412-1422
Berkmen M. (2012) Production of disulfide-bonded proteins in Escherichia coli. Protein Expression and Purification 82:240-251
Vincentell R. and Romier C. (2013) Expression in Escherichia coli: becoming faster and more complex. Current Opinion in Structural Biology 23:326–334
Jia B. and Jeon C.K. (2016) High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. Open Biol. 6:160-196
For proteins that are difficult to express in E. coli, insect cells provide a good alternative. We use baculovirus-mediated expression in lepidopteran insect cell lines. The cell lines we have available in the facility are Sf9, Sf21 and Hi5 cells. For generating the recombinant baculovirus DNA, we use the Bac-to-Bac system from Thermo.
The Baculoviridae are a family of pathogenic insect viruses. The baculovirus most commonly used for recombinant protein expression is Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), which infects species from the Lepidoptera order (moths, butterflies). To understand how baculovirus-mediated recombinant protein production in insect cells works exactly, it’s important to know a little bit about the biology behind it. The baculovirus gene expression occurs in several phases. The early genes are transcribed by the host RNA polymerase II, which means these promoters could be used for recombinant protein production in insect cells. Unfortunately, these promoters are not as strong as some of the promoters from the late and very late genes, which get transcribed by a viral RNA polymerase. Some immediate early promoters such as AcMNPV IE1, OpMNPV OpIE1 and OpIE2 are used in virus-free recombinant protein expression. However, in most cases the strong very late polyhedrin (polH) and p10 promoters are exploited for baculovirus-mediated recombinant protein expression.
The two most commonly used technologies for generating the recombinant baculovirus are the transposition-based method and a method based on homologous recombination in insect cells.
EMBL PEPCF participated in a worldwide benchmarking study that aimed to compare various baculovirus-mediated expression methods used in different lab. The results of this study were published in the Journal of Structural Biology. Interestingly, this benchmarking project showed a 2-fold higher performance of the transposition-based gene integration group and also showed a clear benefit of using v-cath/chiA gene deleted versions of the bacmid backbone such as found in E. coli DH10EMBacY cells. This is also the method we use in the facility to generate all of our recombinant baculoviruses. For our services, we therefore accept all constructs that have plasmid backbone compatible with the Bac-to-Bac transposition-based method for preparing the recombinant baculovirus.
The services we offer at EMBL PEPCF regarding baculovirus-mediated expression in insect cells are the following:
van Oers M.M., Pijlman G.P. and Vlak J.M. (2015) Thirty years of baculovirus–insect cell protein expression: from dark horse to mainstream technology. Journal of General Virology 96:6-23
Clem R.J. and Passarelli A.L. (2013) Baculoviruses: Sophisticated Pathogens of Insects. PLOS Pathogens 9(11): e1003729
Berger I. and Poterszmann A. (2015) Baculovirus expression: old dog, new tricks. Bioengineered 6(6): 316-322
Bleckmann M., Schürig M., Endres M., Samuels A., Gebauer D., Konisch N. and van den Heuvel J. (2019) Identifying parameters to improve the reproducibility of transient gene expression in High Five cells. PLoS One 14(6):e0217878
Bleckmann M., Schürig M., Chen F.F., Yen Z.Z., Lindemann N., Meyer S., Spehr J. and van den Heuvel J. (2016) Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells. PLoS One 11(3):e0149424.
Bleckmann M., Fritz M.H., Bhuju S., Jarek M., Schürig M., Geffers R., Benes V., Besir H. and van den Heuvel J. (2015) Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda. PLoS One 10(8):e0132898
PEPCF provides a service for the transient expression of proteins in mammalian suspension cultures (HEK293F cells) using either standard high-density plasmid transfection protocols or baculovirus-mediated expression in mammalian cells (BacMam).
The services we offer at EMBL PEPCF regarding transient protein expression in mammalian cells are the following: