title: 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 p85alpha, successfully identifying soluble and highly expressed constructs encapsulating all the known globular domains, and immediately suitable for downstream applications.
authors.affiliation: School of Crystallography, Birkbeck College, London WC1E 7HX, United Kingdom.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Reich
authors.forename: Loretto H
authors.initials: LH
authors.lastname: Puckey
authors.forename: Caroline L
authors.initials: CL
authors.lastname: Cheetham
authors.forename: Richard
authors.initials: R
authors.lastname: Harris
authors.forename: Ammar A E
authors.initials: AA
authors.lastname: Ali
authors.forename: Uma
authors.initials: U
authors.lastname: Bhattacharyya
authors.forename: Kate
authors.initials: K
authors.lastname: Maclagan
authors.forename: Keith A
authors.initials: KA
authors.lastname: Powell
authors.forename: Chrisostomos
authors.initials: C
authors.lastname: Prodromou
authors.forename: Laurence H
authors.initials: LH
authors.lastname: Pearl
authors.forename: Paul C
authors.initials: PC
authors.lastname: Driscoll
authors.forename: Renos
authors.initials: R
authors.lastname: Savva
mesh_terms: D020650:Combinatorial Chemistry Techniques; D015723:Gene Library; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012995:Solubility
chemical_list: D010446:Peptide Fragments; D011506:Proteins