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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
reconstitution.
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.
Present studies are directed at increasing the number of coexpressed
subunits and increasing the flexibility of the system.
The VHL-elonginB-elonginC overexpression system useful for hypoxia research is available for licensing to commercial interests. Please contact James Kolonay at the Penn State Intellectual Property Office or Song Tan for more information.
Reference:
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) |
