A major challenge facing structural biologists interested in multicomponent protein complexes is how to isolate large quantities of functional protein complexes. We have developed modular polycistronic expression systems for producing protein complexes in E. coli. The first generation pST39 polycistronic expression system enabled the coexpression of up to 4 protein subunits from a single plasmid.
The second generation pST44 polycistronic expression system improves the modularity of the system and includes a library of affinity tags for efficient purification of the recombinant complexes. We and other laboratories have used these polycistronic expression system to produce milligrams of functionally active prokaryotic and eukaryotic protein complexes containing 2, 3 and 4 subunits, with individual subunit molecular weights of 10-100 kDa. All our biochemical and structural studies of protein complexes have employed the pST39 or pST44 polycistronic expression systems.
The pST39 and pST44 polycistronic expression system plasmids are available from Addgene:
pST39 and pST44 polycistronic expression plasmid suite
You can download the plasmid maps as a pdf document and the plasmid sequences as a text file.
We are grateful for funding support from the NIH National Institute of General Medical Sciences and past support from the Pew Scholars Program in the Biomedical Sciences.
Publications
- Selleck, W. and S. Tan (2008) Recombinant protein complex expression in E. coli, Curr Protocols Protein Sci, Chapter 5, unit 5.21. (abstract)
- Tan, S., R.C. Kern and W. Selleck (2005) The pST44 polycistronic expression system for producing protein complexes in Escherichia coli, Prot. Expr. Purif., 40:385-395. (abstract)
- Barrios, A., W. Selleck, B. Hnatkovich, R. Kramer, D. Sermwittayawong and S. Tan (2007) Expression and purification of recombinant yeast Ada2/Ada3/Gcn5 and Piccolo NuA4 histone acetyltransferase complexes, Methods, 41:271-277. (abstract)
- Lichty, J.L., J.L. Malecki, H.D. Agnew, D.J. Michelson-Horowitz and S. Tan (2005) Comparison of Affinity Tags for Protein Purification, Prot. Expr. Purif., 41:98-105. (abstract)
- Tan, S. (2001) A Modular Polycistronic Expression System for Overexpressing Protein Complexes in Escherichia Coli, Prot. Expr. Purif., 21: 224-234. (abstract)