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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.

SUBMITTER: Reich S

PROVIDER: S-EPMC2242398 | biostudies-literature | 2006 Oct

REPOSITORIES: biostudies-literature

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

Reich Stefanie S Puckey Loretto H LH Cheetham Caroline L CL Harris Richard R Ali Ammar A E AA Bhattacharyya Uma U Maclagan Kate K Powell Keith A KA Prodromou Chrisostomos C Pearl Laurence H LH Driscoll Paul C PC Savva Renos R

Protein science : a publication of the Protein Society 20061001 10

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 b ...[more]

PMID: 17008718

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Project description:We present a novel method utilizing "saltless" pH gradient weak cation exchange-hydrophilic interaction liquid chromatography directly coupled to electron transfer dissociation (ETD) mass spectrometry for the automated on-line high throughput characterization of hypermodified combinatorial histone codes. This technique, performed on a low resolution mass spectrometer, displays an improvement over existing methods with an approximately 100-fold reduction in sample requirements and analysis time. The scheme presented is capable of identifying all of the major combinatorial histone codes present in a sample in a 2-h analysis. The large N-terminal histone peptides are eluted by the pH and organic solvent weak cation exchange-hydrophilic interaction liquid chromatography gradient and directly introduced via nanoelectrospray ionization into a benchtop linear quadrupole ion trap mass spectrometer equipped with ETD. Each polypeptide is sequenced, and the modification sites are identified by ETD fragmentation. The isobaric trimethyl and acetyl modifications are resolved chromatographically and confidently distinguished by the synthesis of mass spectrometric and chromatographic information. We demonstrate the utility of the method by complete characterization of human histone H3.2 and histone H4 from butyrate-treated cells, but it is generally applicable to the analysis of highly modified peptides. We find this methodology very useful for chromatographic separation of isomeric species that cannot be separated well by any other chromatographic means, leading to less complicated tandem mass spectra. The improved separation and increased sensitivity generated novel information about much less abundant forms. In this method demonstration we report over 200 H3.2 forms and 70 H4 forms, including forms not yet detected in human cells, such as the remarkably highly modified histone H3.2 K4me3K9acK14acK18acK23acK27acK36me3. Such detail provided by our proteomics platform will be essential for determining how histone modifications occur and act in combination to propagate the histone code during transcriptional events and could greatly enable sequencing of the histone component of human epigenomes.

Dataset Information

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

Publications

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

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