The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein–ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community.     

Affinity labeling of highly hydrophobic integral membrane proteins for proteome-wide analysis

The ability to identify and quantitate integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and facilitate derivatization of these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified, of which 40 were integral membrane proteins containing from one to nine mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g., ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g., pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses.     

Self-assembly of proteins and their nucleic acids

We have developed an artificial protein scaffold, herewith called a protein vector, which allows linking of an in-vitro synthesised protein to the nucleic acid which encodes it through the process of self-assembly. This protein vector enables the direct physical linkage between a functional protein and its genetic code. The principle is demonstrated using a streptavidin-based protein vector (SAPV) as both a nucleic acid binding pocket and a protein display system. We have shown that functional proteins or protein domains can be produced in vitro and physically linked to their DNA in a single enzymatic reaction. Such self-assembled protein-DNA complexes can be used for protein cloning, the cloning of protein affinity reagents or for the production of proteins which self-assemble on a variety of solid supports. Self-assembly can be utilised for making libraries of protein-DNA complexes or for labelling the protein part of such a complex to a high specific activity by labelling the nucleic acid associated with the protein. In summary, self-assembly offers an opportunity to quickly generate cheap protein affinity reagents, which can also be efficiently labelled, for use in traditional affinity assays or for protein arrays instead of conventional antibodies.     

Protein engineering to optimize recombinant protein purification

Genetic approaches have been used to facilitate purification of recombinant proteins, on both a large and a small scale. Based on developments in three different areas: (i) affinity chromatography; (ii) specific cleavage of fusion proteins and (iii) secretion of fusion proteins, a coupled expression/secretion system was designed. It was further improved by protein engineering. Using a synthetic DNA fragment, encoding two IgG-binding domains derived from staphylococcal protein A, gene products were secreted to the culture medium of Escherichia coli and purified with a one-step affinity procedure. The system has been used for large-scale production of biologically active human peptide hormones, to generate peptides for antibody production and to immobilize proteins on solid supports.     

Overexpression and purification of folded domain of prostate cancer related proteins MSMB and PSA

Overexpression of domains of a human protein using recombinant DNA technology has been challenging because individual domains intend to accumulate as non-soluble aggregate when expressed separately. Studies on identifying right sequences for a domain to be able to fold independently may help understand the folding pattern and underlying protein-engineering events to isolate the functional domains of a protein. In this report, individual domains of prostate cancer related biomarkers; MSMB and PSA were overexpressed in bacterial system and purified in their folded forms using affinity chromatography. The western blotting experiment using domain specific antibodies further confirmed these proteins. The designed nucleotide sequences domains were truncated using fold index software and folding were predicted by phyre2 and I-TASSER software. Other parameters were optimized for their overexpression and purification using Co-NTA affinity chromatography. Purified domains of each protein showed secondary structures such as α + β type for PSA, α/β and β type for the each domains of PSA and MSMB respectively. This is the first report on producing PSA and MSMB individual domains in functional folded forms. This study may help produce the folded domain of many such proteins to be used for better diagnostic purpose.     

Expression and purification of an antitumor-analgesic peptide from the venom of Mesobuthus martensii Karsch by small ubiquitin-related modifier fusion in Escherichia coli

To prevent protein aggregation, some proteins are usually expressed as fusion proteins from which target proteins can be released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the antitumor-analgesic peptide from the venom of Buthus martensii (Karsch) scorpion (AGAP). The optimal expression level of the soluble fusion protein, SUMO-AGAP, was up to 40% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease (Ulp1) to obtain the recombinant AGAP (rAGAP), which was further purified by Ni-NTA affinity chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 7142.63 Dalton, which equaled the theoretically expected mass. N-terminal sequencing of rAGAP showed the sequence corresponded to the native protein. MTT assay indicated the rAGAP could significantly inhibit the proliferation of Jurkat and Hut 78 T lymphoma cell lines. The further writhing experiment showed that the rAGAP had an intensive analgesic effect. The expression strategy presented in this study allows convenient high yield and easy purification of the rAGAP with native sequences.     

A Universal Approach to Prepare Reagents for DNA-Assisted Protein Analysis

The quality of DNA-labeled affinity probes is critical in DNA-assisted protein analyses, such as proximity ligation and extension assays, immuno-PCR, and immuno-rolling circle amplification reactions. Efficient, high-performance methods are therefore required for isolation of pure conjugates from reactions where DNA strands have been coupled to antibodies or recombinant affinity reagents. Here we describe a universal, scalable approach for preparing high-quality oligonucleotide-protein conjugates by sequentially removing any unconjugated affinity reagents and remaining free oligonucleotides from conjugation reactions. We applied the approach to generate high-quality probes using either antibodies or recombinant affinity reagents. The purified high-grade probes were used in proximity ligation assays in solution and in situ, demonstrating both augmented assay sensitivity and improved signal-to-noise ratios.     

Artificial, non-antibody binding proteins for pharmaceutical and industrial applications

Using combinatorial chemistry to generate novel binding molecules based on protein frameworks ('scaffolds') is a concept that has been strongly promoted during the past five years in both academia and industry. Non-antibody recognition proteins derive from different structural families and mimic the binding principle of immunoglobulins to varying degrees. In addition to the specific binding of a pre-defined target, these proteins provide favourable characteristics such as robustness, ease of modification and cost-efficient production. The broad spectrum of potential applications, including research tools, separomics, diagnostics and therapy, has led to the commercial exploitation of this technology by various small- and medium-sized companies. It is predicted that scaffold-based affinity reagents will broaden and complement applications that are presently covered by natural or recombinant antibodies. Here, we provide an overview on current approaches in the biotech industry, considering both scientific and commercial aspects.     

Localization of the Single-Stranded RNA-Binding Domains of Bluetongue Virus Nonstructural Protein NS2

The S2 gene of bluetongue virus, serotype 17, has been cloned, and the nonstructural protein NS2 has been expressed. Synthetic peptides matching regions within the amino acid sequence of NS2 were used to map three single-stranded RNA (ssRNA)-binding regions within the protein. A prokaryotic expression system was then used to generate a series of deletion mutants with the ssRNA-binding domains of NS2 removed, singly and in different combinations. These truncated proteins were expressed on a large scale and purified to near homogeneity. The affinity of each truncated protein towards ssRNA was then assayed by electrophoretic mobility shift assays. As a result, the three ssRNA-binding domains of BTV nonstructural protein NS2 have been conclusively localized, and removal of these three domains completely abrogates the ability of NS2 to bind to ssRNA.     

Development of a compact anti-BAFF antibody in Escherichia coli

Recombinant antibodies, especially single-chain antibody fragment (scFv), can be applied as detection reagents and even substitute for some reagents used in immunoassays. For scFv fragments, there is no such universal system available up to now. We have constructed vectors for the convenient, rapid expression of a novel compact antibody composed of anti-B-cell-activating factor of the TNF family (BAFF) scFv and the Fc portion (the hinge region, CH2, and CH3 domains) of the human IgG1 in Escherichia coli. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The scFv-Fc antibody was demonstrated to retain high binding affinity to antigen, including membrane-bound BAFF and soluble BAFF, and to possess some human IgG crystallizable fragment domain functions, such as human complement C1q and protein A binding. Both size-exclusion high-performance liquid chromatography column analysis and Western blotting of proteins subjected to nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested that scFv-Fc antibody is homodimeric with relative molecular mass of 110 kDa. These findings suggest that the compact antibody may be useful in diagnostic application for the prediction of BAFF relevant to autoimmune diseases in human.     

Novel and economical purification of recombinant proteins: intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association

This work combines two well-established technologies to generate a breakthrough in protein production and purification. The first is the production of polyhydroxybutyrate (PHB) granules in engineered strains of Escherichia coli. The second is a recently developed group of self-cleaving affinity tags based on protein splicing elements known as inteins. By combining these technologies with a PHB-specific binding protein, a self-contained protein expression and purification system has been developed. In this system, the PHB-binding protein effectively acts as an affinity tag for desired product proteins. The tagged product proteins are expressed in E. coli strains that also produce intracellular PHB granules, where they bind to the granules via the PHB-binding tag. The granules and attached proteins can then be easily recovered following cell lysis by simple mechanical means. Once purified, the product protein is self-cleaved from the granules and released into solution in a substantially purified form. This system has been successfully used at laboratory scale to purify several active test proteins at reasonable yield. By allowing the bacterial cells to effectively produce both the affinity resin and tagged target protein, the cost associated with the purification of recombinant proteins could be greatly reduced. It is expected that this combination of improved economics and simplicity will constitute a significant breakthrough in both large-scale production of purified proteins and enzymes and high-throughput proteomics studies of peptide libraries.     

LBT/PTD dual tagged vector for purification, cellular protein delivery and visualization in living cells

Cellular protein delivery is an emerging technique, by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an E. coli expression vector suited for protein cellular delivery experiments. The plasmid is designed to generate a C-terminal fusion with the 12 amino acid HIV-Tat peptide as a protein transduction domain (PTD), whereas the protein N-terminus is fused to an 17-residue peptide lanthanide-binding tag (LBT). LBT is used for both purification by affinity chromatography and fluorescent detection with Tb(3+) as a coordinating metal. We have employed the TA-cloning site between the two tags, LBT and PTD, according to the PRESAT-vector methodology [N. Goda, T. Tenno, H. Takasu, H. Hiroaki, M. Shirakawa, The PRESAT-vector: asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics, Protein Sci. 13 (2004) 652-658], which facilitates unidirectional cloning of any PCR-amplified DNA fragments corresponding to the protein of interest. A simple three-step protocol consisting of affinity purification of LBT/PTD dual-tagged proteins has also been developed, in which the proteins are purified by heparin-, then immobilized Ni(2+)-, and then heparin-affinity chromatography, in this order. The purified protein is ready for protein delivery experiment, and the delivered protein is visible by fluorescent microscopy. Our LBT/PTD dual-tagged PRESAT-vector provides a powerful research tool for exploring cellular functions of proteins in the post-genomic era.     

Functional characterisation of ganglioside-induced differentiation-associated protein 1 as a glutathione transferase

Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene have been linked with Charcot-Marie-Tooth (CMT) disease. This protein, and its paralogue GDAP1L1, appear to be structurally related to the cytosolic glutathione S-transferases (GST) including an N-terminal thioredoxin fold domain with conserved active site residues. The specific function, of GDAP1 remains unknown. To further characterise their structure and function we purified recombinant human GDAP1 and GDAP1L1 proteins using bacterial expression and immobilised metal affinity chromatography. Like other cytosolic GSTs, GDAP1 protein has a dimeric structure. Although the full-length proteins were largely insoluble, the deletion of a proposed C-terminal transmembrane domain allowed the preparation of soluble protein. The purified proteins were assayed for glutathione-dependent activity against a library of 'prototypic' GST substrates. No evidence of glutathione-dependent activity or an ability to bind glutathione immobilised on agarose was found.     

The application of modular protein domains in proteomics

The ability of modular protein domains to independently fold and bind short peptide ligands both in vivo and in vitro has allowed a significant number of protein-protein interaction studies to take advantage of them as affinity and detection reagents. Here, we refer to modular domain based proteomics as "domainomics" to draw attention to the potential of using domains and their motifs as tools in proteomics. In this review we describe core concepts of domainomics, established and emerging technologies, and recent studies by functional category. Accumulation of domain-motif binding data should ultimately provide the foundation for domain-specific interactomes, which will likely reveal the underlying substructure of protein networks as well as the selectivity and plasticity of signal transduction.     

Von Willebrand Factor Type A domain of hCLCA1 is sufficient for U-937 macrophage activation

The human hCLCA1 gene is a member of the CLCA gene family that has a well-documented role in inflammatory airway diseases. Previously, we demonstrated that secreted hCLCA1 plays a role in regulating the innate immune response by activating airway macrophages. However, the mechanism of this regulation remains unclear. In this present study, recombinant proteins containing different hCLCA1 domains are expressed to determine the specific hCLCA1 domain(s) responsible for macrophage activation. Specifically, hCLCA1 constructs containing the hydrolase domain (HYD), the von Willebrand Factor Type A (VWA) domain, and the fibronectin type III (FN3) domain were heterologously expressed and affinity purified through fast protein liquid chromatography. Circular dichroism spectroscopy revealed that the purified hCLCA1 constructs exhibited secondary structure consistent with folded proteins. The VWA domain clearly demonstrated an ability to activate macrophages, inducing an increase in both IL-1β mRNA and protein expression. This activation was associated with the activation of MAPKs and NF-κB pathways, identifying potential mechanistic pathways by which hCLCA1's VWA domain exerts its signaling effect. Altogether, this work identifies a domain with signaling function within hCLCA1, providing a specific target to one of the most highly induced gene products of airway inflammatory disease.     

Cullin 3 Recognition Is Not a Universal Property among KCTD Proteins

Cullin 3 (Cul3) recognition by BTB domains is a key process in protein ubiquitination. Among Cul3 binders, a great attention is currently devoted to KCTD proteins, which are implicated in fundamental biological processes. On the basis of the high similarity of BTB domains of these proteins, it has been suggested that the ability to bind Cul3 could be a general property among all KCTDs. In order to gain new insights into KCTD functionality, we here evaluated and/or quantified the binding of Cul3 to the BTB of KCTD proteins, which are known to be involved either in cullin-independent (KCTD12 and KCTD15) or in cullin-mediated (KCTD6 and KCTD11) activities. Our data indicate that KCTD6^BTB and KCTD11^BTB bind Cul3 with high affinity forming stable complexes with 4:4 stoichiometries. Conversely, KCTD12^BTB and KCTD15^BTB do not interact with Cul3, despite the high level of sequence identity with the BTB domains of cullin binding KCTDs. Intriguingly, comparative sequence analyses indicate that the capability of KCTD proteins to recognize Cul3 has been lost more than once in distinct events along the evolution. Present findings also provide interesting clues on the structural determinants of Cul3-KCTD recognition. Indeed, the characterization of a chimeric variant of KCTD11 demonstrates that the swapping of α2β3 loop between KCTD11^BTB and KCTD12^BTB is sufficient to abolish the ability of KCTD11^BTB to bind Cul3. Finally, present findings, along with previous literature data, provide a virtually complete coverage of Cul3 binding ability of the members of the entire KCTD family.     

Evidence for Differential Binding of Growth Hormones to Membrane and Cytosolic GH Binding Proteins of Rabbit Liver

Abstract

The existence of three GH binding proteins in rabbit liver membranes has been adduced from binding studies with a panel of monoclonal antibodies (1)˙ Immunologically cross-reactive analogues of ‘type 2’ binding proteins were shown to exist in rabbit liver cytosol and in affinity purified receptor from liver microsomes. We now report differences in the binding of human and ovine GH with respect to two antigenic determinants on the ‘type 1″ GH binding protein. The discovery of these differences has enabled the detection of cross-reactive analogues of both binding protein types ‘1″ and ‘2’ in liver cytosol and in affinity purified preparations from liver membranes. These findings show a) a close structural relationship between the pool of cytosolic GH binding proteins and those present in the membranes; and b) differential ligand binding to, as well as absolute ligand selection by GH binding proteins, which could reflect the ability of GH to trigger a range of biological responses either through different receptors or differential interaction with particular receptor subtypes.