A major challenge facing structural biologists interested in multicomponent
protein complexes is how to isolate milligram quantities of functional
protein complexes. In vitro reconstitution of individually expressed
and purified protein components is a common, but time-consuming
and inefficient process. We are studying in vivo reconstitution
by polycistronic expression in E. coli as an alternative to in vitro
We have developed a modular polycistronic expression system for
producing protein complexes in E. coli. The first generation pST39 polycistronic
expression system enables the coexpression of up to 4 protein subunits
from a single plasmid. We have used this polycistronic expression
system to produce milligrams of functionally active yeast and human
protein complexes containing 2, 3 and 4 subunits, with individual
subunit molecular weights of 10-80 kDa.
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.
The pST39 and pST44 polycistronic expression system plasmids are available from Addgene:
You can download the plasmids maps as a pdf document (650 KB) and the plasmid sequences as a txt file (246 KB).
Present studies are directed at increasing the number of coexpressed
subunits and increasing the flexibility of the system.
Selleck, W. and S. Tan (2008) Recombinant protein complex expression in E. coli, Curr Protocols Protein Sci, Chapter 5, unit 5.21. (abstract)
Barrios, A., Selleck, W., Hnatkovich. B., Kramer, R., Sermwittayawong, D. and S. Tan (2007) Expression and purification of recombinant yeast Ada2/Ada3/Gcn5 and Piccolo NuA4 histone acetyltransferase complexes, Methods, 41:271-277. (abstract)
Tan, S., Kern, R.C. and Selleck, W. (2005) The pST44 polycistronic expression system for producing protein complexes in Escherichia coli, Prot. Expr. Purif., 40:385-395. (abstract)
Lichty, J.L., Malecki, J.L., Agnew, H.D., Michelson-Horowitz, D.J. and Tan, S. (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)