Identification of a retina-specific MEKA protein as a 33 K protein

A photoreceptor-specific MEKA protein was purified from bovine retinal soluble fraction. The purified sample was eluted as a single peak of 74 kDa protein from a Superose column, which was dissolved into three components, MEKA protein (32 kDa), beta-(36 kDa) and gamma-(10 kDa) subunits of transducin on a SDS-PAGE. From several lines of evidence, we concluded that MEKA protein is identical with a 33k phosphoprotein reported by Lee et al (1).     

Refolding of a membrane protein in a microfluidics reactor

Membrane protein production for structural studies is often hindered by the formation of non-specific aggregates from which the protein has to be denatured and then refolded to a functional state. We developed a new approach, which uses microfluidics channels, to refold protein correctly in quantities sufficient for structural studies. Green fluorescent protein (GFP), a soluble protein, and bacteriorhodopsin (BR), a transmembrane protein, were used to demonstrate the efficiency of the process. Urea-denatured GFP refolded as the urea diffused away from the protein, forming in the channel a uniform fluorescent band when observed by confocal microscopy. Sodium dodecyl sulphate-denatured BR refolded within the channel on mixing with detergent–lipid mixed micelles. The refolding, monitored by absorbance spectroscopy, was found to be flow rate dependent. This potential of microfluidic reactors for screening protein-folding conditions and producing protein would be particularly amenable for high-throughput applications required in structural genomics. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of a tomato protein that inhibits a xyloglucan-specific endoglucanase

A basic, 51 kDa protein was purified from suspension-cultured tomato and shown to inhibit the hydrolytic activity of a xyloglucan-specific endoglucanase (XEG) from the fungus Aspergillus aculeatus. The tomato (Lycopersicon esculentum) protein, termed XEG inhibitor protein (XEGIP), inhibits XEG activity by forming a 1 : 1 protein:protein complex with a Ki approximately 0.5 nm. To our knowledge, XEGIP is the first reported proteinaceous inhibitor of any endo-beta-1,4-glucanase, including the cellulases. The cDNA encoding XEGIP was cloned and sequenced. Database analysis revealed homology with carrot extracellular dermal glycoprotein (EDGP), which has a putative role in plant defense. XEGIP also has sequence similarity to ESTs from a broad range of plant species, suggesting that XEGIP-like genes are widely distributed in the plant kingdom. Although Southern analysis detected only a single XEGIP gene in tomato, at least five other XEGIP-like tomato sequences have been identified. Similar small families of XEGIP-like sequences are present in other plants, including Arabidopsis. XEGIP also has some sequence similarity to two previously characterized proteins, basic globulin 7S protein from soybean and conglutin gamma from lupin. Several amino acids in the XEGIP sequence, notably 8 of the 12 cysteines, are generally conserved in all the XEGIP-like proteins we have encountered, suggesting a fundamental structural similarity. Northern analysis revealed that XEGIP is widely expressed in tomato vegetative tissues and is present in expanding and maturing fruit, but is downregulated during ripening.     

Identification of a member of a new RNase a family specifically secreted by epididymal caput epithelium

In this study, we purified the first member of a new ribonuclease (RNase) A family from fluid of the proximal caput of the boar epididymis. This protein, named "Train A," is the most abundant compound secreted in the anterior part of the boar epididymis. After 2D electrophoresis, it is characterized by more than 10 isoforms ranging in size from 26 to 33 kDa and pI from 5 to 8.5. Several tryptic peptides were N-terminal sequenced, and an antiserum against one of these peptides was obtained. The protein was immunolocalized in the epididymal epithelium of the proximal caput, especially in the Golgi zone and the apical cytoplasm of the principal cells. In the lumen, spermatozoa were negative but droplets of reaction product were observed within the lumen. Full lengths of Train A cDNA were obtained from a lambdagt11 boar caput epididymis library and sequenced. The deduced protein is composed of 213 amino acids, including a 23-amino acid peptide signal and a potential N-glycosylation site. The mRNA of this protein has been retrieved and partially sequenced in the bull, horse, and ram, and homologous cDNA is found in databanks for the rat, mouse, and human. All the sequences are highly conserved between species. This protein and its mRNA are male-specific and exclusively expressed in the proximal caput of the epididymis, the only site where they have been found. Train A presents an RNase A family motif in its sequence. The RNase A family is a group of several short proteins (20-14 kDa) with greater and lesser degrees of ribonucleolytic activity and with supposed different roles in vivo. However, the presence of a long-conserved N-terminal specific sequence and the absence of RNase catalytic site for Train A indicate that Train A protein is a member of a new family of RNase A.     

Characterization of a yeast phase specific protein from a fungus, Histoplasma capsulatum

We have characterized an unusual yeast phase specific protein from Histoplasma capsulatum. The protein, which we have called protein 6, is produced by the yeast cells which have been derepressed for sulfite reductase, and it can account for more than 40% of the total extract protein. Synthesis of both sulfite reductase and protein 6 is subject to cysteine repression. However, sulfite reductase activity is maximal in logarithmically growing cells whereas protein 6 is synthesized de novo and accumulated by stationary phase cells. The following are the major physicochemical properties of protein 6: (1) the native protein has a molecular weight of about 15 000; (2) electrophoresis on a sodium dodecyl sulfate polyacrylamide gel yielded a single band with a molecular weight 7600; (3) protein 6 is capable of reducing the dye, nitroblue tetrazolium, and cytochrome c, a property that has been found to be shared by a number of trypsin inhibitors, and (4) the molecule is negatively charge and is relatively resistant to proteolysis. The amino acid composition of protein 6 has been determined.     

Identification of a novel protein phosphatase catalytic subunit by cDNA cloning

A cDNA encoding a novel protein phosphatase catalytic subunit (protein phosphatase X) has been isolated from a rabbit liver library. It codes for a protein having 45% and 65% amino acid sequence identity, respectively, to the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A from skeletal muscle. The enzyme is neither the hepatic form of protein phosphatase 1 or 2A, nor is it protein phosphatase 2B or 2C. The possible identity of protein phosphatase X is discussed.     

Identification of a transformation-specific protein induced by a Rous sarcoma virus.

Using antisera obtained from rats bearing Schmidt-Ruppin strain Rous sarcoma virus-induced tumors, we have idnetified a protein with an apparent molecular weight of 56,000 daltons and an isoelectric point of 6.3 in extracts of chick embryo fibroblasts transformed by a wild-type nondefective Rous sarcoma virus (Schmidt-Ruppin strain). This protein was not found in cells infected by trnasformation-defective mutants with either a partial or complete deletion of the src gene, nor in cells infected by a nontransforming avian leukosis virus. The 56,000 dalton molecular weight protein was found to be synthesized at both the permissive and nonpermissive temperatures in cells infected by either of two conditionallethal mutants that are temperature-sensitive in cell transformation. The amount of this protein, however, accumulated in cells infected by these temperature-sensitive mutants, relative to the structural polypeptides, differed significnatly from that seen with the nondefective virus. Pulsechase experiments indicate that the protein is extremely unstable, with a half-life of about 20 min, and does not serve as a precursor to any of the detectable virion polypeptides. Furthermore, incubation of the rat antiserum with purified, disrupted virus did not affect its immunoreactivity to this particular protein. We conclude that this 56,000 dalton molecular weight protein is a nonstructural protein specific to cells transformed by Rous sarcoma virus.     

The ram: a novel low molecular weight GTP-binding protein cDNA from a rat megakaryocyte library

A novel low Mr GTP-binding protein cDNA was isolated from a rat megakaryocyte cDNA library with a synthetic oligonucleotide probe corresponding to an 8-amino acid sequence specific for c25KG, a GTP-binding protein previously isolated from human platelet cytosol fraction [(1989) J. Biol. Chem. 264, 17000-17005]. The cDNA has an open reading frame encoding a protein of 221 amino acids with a calculated Mr of 25068. The protein is designated as ram (ras-related gene from megakaryocyte) protein (ram p25). The amino acid sequence deduced from the ram cDNA contains the consensus sequences for GTP-binding and GTPase domains. ram p25 shares about 23%, 39% and 80% amino acid homology with the H-ras, smg25A and c25KG proteins, respectively. The 3.5-kb ram mRNA was detected abundantly in spleen cells.     

Identification of a single-copy gene encoding a Type I chlorophyll a/b-binding polypeptide of photosystem I in Arabidopsis thaliana

We have isolated and sequenced cDNA and genomic clones from Arabidopsis thaliana which specify a 241 residue protein with 84% sequence identity to a photosystem I Type I chlorophyll a/b -binding (CAB) protein from tomato. The open reading frame is interrupted by three introns which are found at equivalent positions as the corresponding introns in the tomato gene. Comparison to the amino acid sequence of other CAB proteins confirms that all CAB proteins share two regions of very high similarity. However, near the N-terminus and between the conserved regions this light-harvesting complex I (LHCI) protein, as other LHCI proteins from other plant species, has sequence motifs which appear to be PSI-specific. Restriction analysis of genomic DNA shows that the Arabidopsis protein is encoded by a single-copy gene.     

Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody

The principal iodinatable surface protein (P30) of our cloned RH strain of Toxoplasma gondii has an apparent molecular weight of 30,000, as measured by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Monoclonal antibody B specifically immunoprecipitated protein P30 from a detergent extract of surface radioiodinated T. gondii. Monoclonal antibody B in the presence of complement was also parasiticidal for T. gondii, and this parasiticidal effect could be blocked by protein P30. Monoclonal antibody B was purified from mouse ascitic fluid and linked to cyanogen bromide-activated Sepharose. The resulting immunoabsorbent was used to purify 1.7 mg of protein P30 from a large number of parasites. The efficiency of recovery of protein P30 was measured by assays of radioactivity and of parasiticidal blocking activity. Protein P30 represented 3 to 5% of the total protein. It is also present in a recently isolated strain of T. gondii. A convalescent human antitoxoplasma serum immunoprecipitated radiolabeled protein P30. Three convalescent antisera when quantitated by an ELISA test had a high anti-protein P30 titer. Charge shift electrophoresis showed that protein P30 has an extensive hydrophobic region and thus is probably an integral membrane protein. Electrophoresis under nonreducing conditions showed no evidence that protein P30 exists as a disulfide linked homo- or heterodimer, although it probably has intramolecular disulfide bonds.     

Extended reading frame of a ubiquitin gene encodes a stable, conserved, basic protein

Antibodies specific for the 80-amino acid hypothetical protein encoded by the in-frame, 3'-extension of a human ubiquitin gene were produced in rabbits by immunization with a 14-residue synthetic peptide. When used to probe HeLa cell extracts for the non-ubiquitin product of this natural fusion gene, the antipeptide sera detected a protein with an apparent molecular weight of 16,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An immunoreactive protein of identical mobility was detected in organisms ranging from Acanthamoeba to man, indicating that the extension protein, like ubiquitin, is highly conserved. The immunoreactive protein was isolated from calf thymus, and direct sequencing revealed the first 16 amino acids to be identical to those predicted from the extension portion of the human cDNA. Thus, ubiquitin was no longer present at the amino terminus. The purified bovine extension protein failed to react with a ubiquitin-specific antibody indicating the absence of isopeptide-linked ubiquitin as well. Moreover, by denaturing gel permeation chromatography the extension has a molecular weight of 10,000 Da, a value that corresponds more closely to the size of the extension alone (9,000 Da) than to the intact fusion protein (17,500 Da). The extension protein, which was found in both cytoplasmic and nuclear fractions of HeLa cells, persisted at high levels when protein synthesis was blocked with cycloheximide or puromycin. These results show that the 80-residue extension protein is the stable, processed product of the ubiquitin fusion gene.     

Molecular cloning and partial characterization of a plant VAP33 homologue with a major sperm protein domain

In a search for proteins interacting with the resistance protein Cf9 from tomato, a new cDNA was cloned and characterized. Protein sequence database searches suggested that the 120 residue-N terminal domain of the encoded protein (named VAP27) is highly similar to the VAP33 protein family from animals, to uncharacterized plant proteins, and to a lower extent, to the major sperm protein (MSP) from nematodes. The second half of the protein is similar to VAMP and to the VAP33 N-terminus comprising a predicted coiled-coil region followed by a transmembrane segment. The sequence/structure comparison of VAP27 with the crystal structure of AsMSP1 from Ascaris suum, using molecular modeling with the threading method, suggested that the N-terminus of VAP27 does possess a MSP-like domain that might participate in the formation of a protein-protein network. The coiled-coil region of VAP27 was modeled based on the structure of the VAP- and VAMP-containing SNARE complex. The coiled-coil region might also be involved in protein-protein interactions similar to VAP-VAMP interactions.     

Severe acute respiratory syndrome coronavirus 3a protein is released in membranous structures from 3a protein-expressing cells and infected cells

Severe acute respiratory syndrome coronavirus (SCoV) accessory protein 3a is a virus structural protein. We demonstrate here that 3a protein was released efficiently in membranous structures from various cell lines expressing 3a protein. A subpopulation of the released 3a protein is associated with detergent-resistant membranes. The presence of the YxxPhi and diacidic motifs, located within the cytoplasmic tail of the 3a protein, was not required for its efficient release. Analysis of supernatant from SCoV-infected cells with sucrose gradient sedimentation and virus capture assay indicated that the 3a protein was released from infected cells in two distinct populations, as a component of SCoV particles, and in membrane structures with a lower buoyant density. These data provide new insights into the biological properties of SCoV 3a protein.     

Molecular characterization of a gene encoding a cysteine-rich protein preferentially expressed in anthers of Lycopersicon esculentum

The tomato (Lycopersicon esculentum Mill.) cDNA clone TomA5B was isolated by differential screening of a cDNA library prepared from anthers at late meiosis to tetrad formation. The 5B gene is present in a single copy in the tomato genome. Expression is developmentally regulated and tissue specific. RNA accumulation was detected from premeiosis through tetrad release in the tapetal cell layer of the anther with low levels of RNA detected in petals and early stages of pistil development. The protein deduced from the DNA sequence analysis is predicted to have a molecular mass of 11.1 kDa and a secretory signal sequence, suggesting it is a secreted protein. The deduced 5B protein has a pattern of cysteine residues that is similar to other proteins that have stamen-specific expression and to a superfamily of seed proteins. The 5B protein is unique in that there is no amino acid sequence similarity to other proteins beyond the similar cysteine motif.     

Electroelution of protein fractions from a polyacrylamide gel

A method for electroelution of protein fractions from polyacrylamide gel and device for performing such a process have been developed. The application of two tris-glycine buffers with the low and high ionic strength, pH 9.0-9.2 provides a concentration of protein simultaneously to extraction from the gel. The duration of elution is in the range of 1-3 hours and depends on the protein mobility. The effectiveness of the system is demonstrated for disc-electrophoretic separation and electrophoresis in slab gel in the presence of SDS. The maximal amount of pure protein fraction obtained is about 4.5-5.0 mg. The method may be useful especially for the fractionation of limited quantities of protein samples.     

Reconstitution of a Mg-ATP-dependent protein phosphatase and its activation through a phosphorylation mechanism

A Mg-ATP-dependent protein phosphatase has been reconstituted from the catalytic subunit of protein phosphatase-1 and inhibitor-2, and consists of a 1:1 complex between these proteins. Activation of this enzyme by glycogen synthase kinase-3 and Mg-ATP results from the phosphorylation of inhibitor-2 on a threonine residue(s) and is accompanied by the dissociation of the complex. The results prove that protein phosphatase-1 and the Mg-ATP-dependent protein phosphatase contain the same catalytic subunit, and that they are interconvertible forms of the same enzyme.     

Thin filaments of molluscan catch muscles contain a calponin-like protein

A novel 40 kDa protein was detected in native thin filaments from catch muscles of the mussel Crenomytilus grayanus. The MALDY-TOF analysis of the protein showed a 40% homology with the calponin-like protein from the muscle of Mytilus galloprovincialis (45 kDa), which has a 36% homology with smooth muscle calponin from chicken gizzard (34 kDa). The amount of the calponin-like protein in thin filaments depends on isolation conditions and varies from the complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the contact of the protein in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22 degrees C and optimal values of both pH and ionic strength of the extraction solution, total calponin-like protein coprecipitates with thin filaments. At 2 degrees C it remains in the supernatant. The 40 kDa calponin-like protein from the mussel Crenomytilus grayanus has similar properties with smooth muscle calponin (34 kDa). It is thermostable and inhibits the actin-activated Mg -ATPase activity of actomyosin. In addition, the 40 kDa calponin-like protein isolated without using thermal treatment contains endogenous kinases. It was found that the calponin-like protein can be phosphorylated by endogenous kinases in the Ca -independent manner. These results indicate that the calponin-like protein from the catch muscle of the mussel Crenomytilus grayanus is a new member of the calponin family. The role of proteins from this family both in muscle and ponmuscle cells is still obscure. We suggest that the calponin-like protein is involved in the Ca -independent regulation of smooth muscle contraction.     

Crystal structure of a protein associated with cell division from Mycoplasma pneumoniae (GI: 13508053): a novel fold with a conserved sequence motif

UPF0040 is a family of proteins implicated in a cellular function of bacteria cell division. There is no structure information available on protein of this family. We have determined the crystal structure of a protein from Mycoplasma pneumoniae that belongs to this family using X-ray crystallography. Structural homology search reveals that this protein has a novel fold with no significant similarity to any proteins of known three-dimensional structure. The crystal structures of the protein in three different crystal forms reveal that the protein exists as a ring of octamer. The conserved protein residues, including a highly conserved DXXXR motif, are examined on the basis of crystal structure.     

Molecular cloning and characterization of a gene encoding a 13.1 kDa antigenic protein of Naegleria fowleri

An antigen-related gene was cloned from a cDNA expression library of Naegleria fowleri by immunoscreening with sera obtained from mice that were either immunized with an amoebic lysate or infected with trophozoites. The coding nucleotide sequence of the cloned gene consisted of 357 bases that were translated into 119 amino acids. This gene was designated as nfa1. The predicted amino acid sequence of Nfa1 protein has two potential glycosylation and three potential phosphorylation sites, and its predicted secondary structure consists of four helices and three corners. The deduced amino acid sequence of Nfa1 protein shares 43% identity with the myohemerythrin (myoHr) protein from a marine annelid, Nereis diversicolor, including 100% identity in conserved regions and iron-binding residues. A phylogenetic tree constructed from amino acid sequences placed the N. fowleri Nfa1 protein outside of a cluster of myoHr proteins from eight invertebrates. A purified recombinant protein that migrated as a 13.1 kDa species in SDS-PAGE was produced. This recombinant protein exhibited a strong immunoreactivity with infected, immune, and anti-Nfal sera. In addition, an anti-Nfa1 serum reacted with an amoeba lysate in immunoblotting analysis. The present nfal gene encoding the myoHr-like protein is the first myoHr gene cloned from protozoa, and the Nfal antigen may be useful in diagnostic studies     

Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), β-mercaptoethanol (β-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.