As a test case, we analysed truncation libraries of the influenza polymerase PB2 subunit where our earlier ESPRIT protocol had proved fruitful in identifying previously unknown domains for structural analyses. Besides providing biologically interesting examples of Adriamycin Topoisomerase inhibitor co-folded and pre-folded subunits, an additional aim was to see whether previously uncharacterised constructs could be identified through expression of validated, potentially stabilising partners. The N terminus of PB2 has been shown recently by X-ray crystallography to co-fold with the C terminus of PB1 whilst the C terminus contains a small domain that supports a NLS that has been shown, also by X-ray crystallography, to bind tightly to importin a nuclear import receptors. Investigations into the domain structures of the influenza polymerase have led to a series of new crystal structures by our lab and others, reviewed in. Here we have identified a series of constructs by empirical screening that form stable complexes with known partners validated recently through structural studies: PB2- importin a and PB1�CPB2. The availability of these structural data permits us to rationalise the results of the screen and the second step of re-expression of hits minus bait protein. Of the PB1�CPB2 complexes identified, the smallest PB1- -PB2- is similar to the ordered residues in a recent crystal structure that shows the two polypeptides to intertwine, forming a structural module. When expressing the subunits separately, the stability of the constructs is severely compromised. In contrast, the C-terminal PB2 constructs can be expressed in the absence of the importin a1 bait with no loss of solubility, consistent with their structural independence and previous studies showing that the proteins can be produced separately and complexes constituted by mixing. In summary, CoESPRIT is an efficientmethod for identification of purifiable soluble complexes at yields compatible with downstream studies. Interacting polypeptides are identified from small scale screening that, following scale up, should be validated by size exclusion chromatography or other biophysical techniques. They can be used directly in some applications, or may require further refinement including limited proteolysis to remove unstructured regions, or tag removal prior to crystallisation trials. However these are standard procedures that are greatly facilitated by the availability of purified complexes as starting material. CoESPRIT, in common with other library methods including our original method employs the principles of directed evolution whereby a target gene encoding a poorly expressed target is incrementally truncated to generate PF-04217903 random genetic diversity.