Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high‐throughput applications

Abstract

Exploitation of potential new targets for drug and vaccine development has an absolute requirement for multimilligram quantities of soluble protein. While recombinant expression of full-length proteins is frequently problematic, high-yield soluble expression of functional subconstructs is an effective alternative, so long as appropriate termini can be identified. Bioinformatics localizes domains, but doesn't predict boundaries with sufficient accuracy, so that subconstructs are typically found by trial and error. Combinatorial Domain Hunting (CDH) is a technology for discovering soluble, highly expressed constructs of target proteins. CDH combines unbiased, finely sampled gene-fragment libraries, with a screening protocol that provides ''holistic'' readout of solubility and yield for thousands of protein fragments. CDH is free of the ''passenger solubilization'' and out-of-frame translational start artifacts of fusion-protein systems, and hits are ready for scale-up expression. As a proof of principle, we applied CDH to p85a, successfully identifying soluble and highly expressed constructs encapsulating all the known globular domains, and immediately suitable for downstream applications.

ORF-selector ESPRIT: A second generation library screen for soluble protein expression employing precise open reading frame selection

“…In our proof-of-principle study, we analysed p85a, a protein comprising five previously characterised domains and which has been used previously in similar work, establishing a benchmark (Reich et al, 2006). In that early CDH study, 1404 clones with p85a DNA inserts were processed through a screening procedure resulting in several purified constructs.…”

“…To further complicate matters, small variations in just a few amino acid residues of the fold edges can dramatically and unpredictably affect the yield, solubility and stability of the final constructs. To address all of these problems simultaneously in a manner described as ''holistic'' (Reich et al, 2006), random library construct screening technologies for identification of soluble protein domains have been developed. Of particular note are combinatorial domain hunting (CDH) (Reich et al, 2006), colony filtration (CoFi) (Cornvik et al, 2006) and expression of soluble proteins by random incremental truncation (ESPRIT) (Tarendeau et al, 2007;Yumerefendi et al, 2010).…”

“…To address all of these problems simultaneously in a manner described as ''holistic'' (Reich et al, 2006), random library construct screening technologies for identification of soluble protein domains have been developed. Of particular note are combinatorial domain hunting (CDH) (Reich et al, 2006), colony filtration (CoFi) (Cornvik et al, 2006) and expression of soluble proteins by random incremental truncation (ESPRIT) (Tarendeau et al, 2007;Yumerefendi et al, 2010). All methods have in common a construct library generation step coupled to a high throughput colony screen for soluble constructs (Hart and Tarendeau, 2006;Prodromou et al, 2007;Savva et al, 2007).…”

“…But is it necessary to screen all the diversity in a library? Clearly several successful examples exist where useful solutions were obtained from undersampled libraries (Bonneau et al, 2009;Guilligay et al, 2008;Reich et al, 2006). However it is a general principle in directed evolution experiments that the larger the library screened, the bigger the phenotype improvement obtained.…”

“…To demonstrate this system, we screened p85a, a regulatory subunit of class 1A phosphoinositide 3-kinase from which a number of domains have been characterised structurally (Booker et al, 1992;Booker et al, 1993;Hoedemaeker et al, 1999;Liang et al, 1996;Miled et al, 2007;Musacchio et al, 1996;Nolte et al, 1996;Siegal et al, 1998). This gene was used in the elucidation of the CDH method (Reich et al, 2006) and would appear to be a good benchmark for this type of method. All domains and many multiple domains were identified as highly expressed purifiable forms.…”