Soluble Protein Domains Research Articles

A limited universe of membrane protein families and folds

One of the goals of structural genomics is to obtain a structural representative of almost every fold in nature. A recent estimate suggests that 70%-80% of soluble protein domains identified in the first 1000 genome sequences should be covered by about 25,000 structures-a reasonably achievable goal. As no current estimates exist for the number of membrane protein families, however, it is not possible to know whether family coverage is a realistic goal for membrane proteins. Here we find that virtually all polytopic helical membrane protein families are present in the already known sequences so we can make an estimate of the total number of families. We find that only approximately 700 polytopic membrane protein families account for 80% of structured residues and approximately 1700 cover 90% of structured residues. While apparently a finite and reachable goal, we estimate that it will likely take more than three decades to obtain the structures needed for 90% residue coverage, if current trends continue.

Self-Assembling Hydrogels

The overview focuses on the design, synthesis, and characterization of peptide/protein-containing polymers, which self-assemble into stimuli-sensitive hydrogels. Hybrid hydrogels are composed of two classes of molecules, synthetic and biological. They self-assemble from graft copolymers, block copolymers and by association of soluble polymers and protein domains. Genetic engineering provides a powerful tool to produce biomaterials (macromolecules) with precise control of composition, length, and three-dimensional structure. Minimal changes in the composition of the coiled-coil forming protein domains in genetically produced triblock copolypeptides result in dramatic changes in the properties of the copolymers and, ultimately, the self-assembled hydrogels. The application of self-assembling hydrogels as biomaterials has been investigated and has shown a promising future.

Bacterial expression strategies for human angiogenesis proteins

We outline an expression strategy using Escherichia coli to obtain soluble components of a selected group of human proteins implicated in angiogenesis. These targets represent a heterogeneous group of proteins which for expression purposes were separated into cytoplasmic and helical membrane protein categories. Target selection was refined using a bioinformatic approach to generate a list of 50 experimental targets. A group consisting of forty-four cytoplasmic and signal-containing protein targets were amplified and cloned into multiple expression vectors. For this target category, we obtained 48% soluble expression products. In addition, we used a domain expression approach for six high molecular weight proteins predicted to contain membrane spanning helices to obtain soluble domain products. These results validate the utility of a bioinformatically driven high throughput approach to increase the number of soluble proteins or protein domains which can be used for multiple downstream applications.

Soluble domains of telomerase reverse transcriptase identified by high‐throughput screening

Telomerase is a ribonucleoprotein complex responsible for extending the ends of eukaryotic chromosomes. Structural and biophysical studies of this enzyme have been limited by the inability to produce large amounts of recombinant protein. Here we perform a high-throughput screen to map regions of the Tetrahymena thermophila TERT (Telomerase Reverse Transcriptase) protein that are overexpressed in a soluble form in Escherichia coli using a GFP-fusion system. Many of the soluble protein domains identified do not coincide with domains inferred from multiple sequence alignment, so screening for fluorescent colonies provided information not otherwise readily obtained. The method revealed an essential, independently folded N-terminal domain that was expressed and purified with high yield and found to be suitable for structural analysis. These results provide a tool for future structural and biophysical studies of TERT.

The PRESAT-vector: asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics.

A rapid unidirectional method for cloning PCR-amplified cDNA fragments into virtually any fusion protein expression vector is described. The method, termed PRESAT-vector cloning, is based on a T-vector technique that does not require restriction endonuclease digestion of the PCR product. Subsequently, we applied a novel ORF selection method of the ligated plasmid products. This second step involves restriction endonuclease treatment that eliminates the plasmids containing an ORF in the wrong orientation prior to transformation into the bacterial host for further protein expression studies. To achieve this selection, we customized the 5'-sequence of the "rear" PCR primer corresponding to the C terminus of the protein to be expressed. The colonies harbored only the ligated products of the desired orientation at >90% efficiency. This method is applied to a GST fusion expression system, and an HTS system for soluble proteins from an expression library was tested.

A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs

Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli. Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E. coli. This system experimentally identified any random cDNA fragments producing soluble protein domains. Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag. These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins. The latter is useful as a fluorescence indicator of protein folding. The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag. This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel.

SUPERFAMILY: HMMs representing all proteins of known structure. SCOP sequence searches, alignments and genome assignments.

The SUPERFAMILY database contains a library of hidden Markov models representing all proteins of known structure. The database is based on the SCOP 'superfamily' level of protein domain classification which groups together the most distantly related proteins which have a common evolutionary ancestor. There is a public server at http://supfam.org which provides three services: sequence searching, multiple alignments to sequences of known structure, and structural assignments to all complete genomes. Given an amino acid or nucleotide query sequence the server will return the domain architecture and SCOP classification. The server produces alignments of the query sequences with sequences of known structure, and includes multiple alignments of genome and PDB sequences. The structural assignments are carried out on all complete genomes (currently 59) covering approximately half of the soluble protein domains. The assignments, superfamily breakdown and statistics on them are available from the server. The database is currently used by this group and others for genome annotation, structural genomics, gene prediction and domain-based genomic studies.

Random PCR-Based Screening for Soluble Domains Using Green Fluorescent Protein

A novel approach to screen soluble protein domains is presented, by combining tagged random primer polymerase chain reaction (PCR) method and protein-folding assay using green fluorescent protein. Tagged random primer PCR method was used to amplify random DNA fragments from a template cDNA. The PCR products were fused to the green fluorescent protein (GFP) gene. Then solubilities of their translation products were rapidly monitored by the fluorescence of transformed Escherichia coli colonies on plates. We succeeded in cloning four soluble domains from Vav protein using this method. The present method is applicable to all proteins when cDNAs are available.

Synthetic competition between cytoplasmic folding and translocation of a soluble membrane protein domain

In wild-type strains of Escherichia coli, alkaline phosphatase (AP), either when present as a soluble protein or when fused to a membrans protein, is only active after translocation to the periplasm. In thioredoxin reductase ( trxB) mutants, however, cytoplasmically localized AP can form disulphide bonds and can reach an active conformation. Once it has folded in the cytoplasm, it can no longer be translocated. On the other hand, when AP is fused to periplasmic domains of a membrane protein, translocation can be more rapid than folding. Thus, expressing hybrids of AP and integral membrane proteins in a trxB mutant generates competition between folding of AP in the cytoplasm and its translocation to the periplasm. The cellular localization of AP can be monitored in phosphoserine phosphatase ( serB) mutants causing auxotrophy for L-serine. Cytoplasmically but not periplasmically localized AP can compensate for the lack of SerB, leading to growth on indicator plates. As expected, when AP was fused to cytoplasmic domains of membrane proteins, serBmediated auxotrophy was abolished. Surprisingly, AP fusions to periplasmic domains exhibited a non-uniform response pattern. Fusions that translocate AP rapidly did not complement the SerB defect while those that export AP only slowly could do so. The usefulness of these strains for studying a variety of aspects related to membrane protein biogenesis is discussed.

Cloning, expression, and immunological characterization of recombinant Lolium perenne allergen Lol p II.

The molecular cloning of the cDNA encoding for an isoallergenic form of Lol p II, a major rye grass (Lolium perenne) pollen allergen, was performed by polymerase chain reaction amplification on mRNA extracted from pollen. The amino acid sequence derived from the cDNA was truncated by 4 and 5 residues at the NH2- and COOH-terminal ends, respectively, and differed only in one position from that previously reported. This cDNA was expressed in Escherichia coli by fusion to the carboxyl terminus of the human ferritin H-chain. The molecule was produced in high yields as a soluble protein and was easily purified. The protein retains the multimeric quaternary structure of ferritin, and it exposes on the surface the allergenic moiety, which can be recognized in Western blotting and in enzyme-linked immunosorbent assay experiments by specific IgE from allergic patients. The recombinant allergen was used to analyze the sera of 26 patients allergic to L. perenne compared with control sera. The results were in good agreement with the values obtained with the radioallergosorbent test assay. In addition, histamine release experiments in whole blood from an allergic patient and skin prick tests showed that the recombinant allergen retains some of the biological properties of the natural compound. These findings indicate that the availability of homogeneous recombinant allergens may be useful for the development of more specific diagnostic and therapeutic procedures. Moreover, this expression system may be of more general interest for producing large amounts of soluble protein domains in E. coli.