Molecular studies of proteins often fail because sufficient quantities of the protein cannot be isolated in a soluble and correctly folded form. This issue is particularly acute for complex and multi-domain proteins that may not express or fold well in the commonly used recombinant expression systems.   To help overcome this issue, we are developing a high throughput method to efficiently screen fragments of a target protein for soluble expression in Escherichia coli.  In this ‘domain seeking’ technique we first generate a random gene fragment library for the target protein using PCR-based or mechanical methods.¹  The gene fragment library is then expressed in E. coli and a split-green fluorescent protein (split-GFP) assay is used to report on protein fragment solubility.²  As part of our initial trials of this technique we successfully identified the two structural domains of a well-characterised viral protein. 

In the past, protein domain boundaries have been identified through partial proteolytic digests of the full-length protein or with bioinformatic analysis through comparison with similar proteins.  The advantage of domain seeking is that it is an ab initio procedure; neither a sample of the full-length protein nor structural information on related proteins is required.  We are now applying domain seeking to a variety of viral, bacterial and human proteins, which are difficult to express recombinantly in a full-length form.

References

1.       Reich, S.; Puckey, L. H.; Cheetham, C. L.; Harris, R.; Ali, A. A.; Bhattacharyya, U.; Maclagan, K.; Powell, K. A.; Prodromou, C.; Pearl, L. H.; Driscoll, P. C.; Savva, R., Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications. Protein science : a publication of the Protein Society 2006, 15, 2356-65.

2.       Cabantous, S.; Terwilliger, T. C.; Waldo, G. S., Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nature biotechnology 2005, 23, 102-7.