Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0276640 (TEM)
 20,729 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A gene, pkn2, encoding a Myxococcus xanthus protein with significant similarities to eukaryotic protein serine/threonine kinases, was cloned using the polymerase chain reaction. The open reading frame for the protein, beginning with a GUG initiation codon, consists of 830 amino acids. The amino-terminal 279 residues show 37% identity to catalytic domain of Pkn1, another protein serine/threonine kinase expressed during the development at the onset of sporulation. The catalytic domain of Pkn2 contains 27% and 25% identity to rat Ca2+/calmodulin-dependent protein kinase and Bos taurus rhodopsin kinase, respectively. In the middle of the carboxy-terminal regulatory domain, there is a typical transmembrane domain consisting of 18 hydrophobic residues. The gene product, Pkn2, produced in Escherichia coli under a T7 promoter was phosphorylated at both serine and threonine residues. TEM-beta-lactamase produced in E. coli was found to serve as an effective substrate for Pkn2, phosphorylated only at threonine residues, shifting its apparent molecular mass from 29 to 44 kD. The phosphorylated beta-lactamase was unable to be secreted into the periplasmic space and localized in the cytoplasmic and membrane fractions. Analysis of phoA fusions with pkn2 demonstrated that Pkn2 is a transmembrane protein with the kinase domain in the cytoplasm and the 207-residue carboxy-terminal domain outside the cytoplasmic membrane. Disruption of pkn2 showed no effect on vegetative growth but reduced the yield of myxospores by 30%-50%. On the basis of the present results, we propose that Pkn2 is a transmembrane protein serine/threonine kinase that regulates the activity of endogenous beta-lactamase or related enzymes in response to an external signal yet to be identified.
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PMID:Myxococcus xanthus, a gram-negative bacterium, contains a transmembrane protein serine/threonine kinase that blocks the secretion of beta-lactamase by phosphorylation. 777 14

Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino-terminal OmpA signal sequence was fused with TEM beta-lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia coli as an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the beta-lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor. In the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal beta-lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino-terminal signal sequence. Introduction of two lysine residues at the carboxy-terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the amino-terminal nuclease domain across the membrane, leaving the carboxyl terminal beta-lactamase domain in the cytoplasm. When three more lysine residues were added to the amino-terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino-terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.
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PMID:Reversible topology of a bifunctional transmembrane protein depends upon the charge balance around its transmembrane domain. 802 60

Overproduction of the third topological domain of the transmembrane protein TolA (TolAIII) in the periplasm of Escherichia coli confers a "leaky" phenotype to host cells by disrupting the integrity of the outer membrane and causing periplasmic proteins to leach into the growth medium. To examine the physiological consequences of TolAIII overexpression in more detail and assess the usefulness of this strategy for the release of periplasmic recombinant proteins into the extracellular fluid, we constructed a ColE1-compatible plasmid encoding a fusion between the ribose binding protein signal sequence and TolAIII under T7lac transcriptional control. About half of the total TolAIII synthesized in IPTG-induced cells aggregated in a precursor form in the cytoplasm. However, the majority of the mature protein was soluble and located in the extracellular fluid. TolAIII-overproducing cultures exhibited only slight growth defects upon entry into stationary phase but underwent extensive lysis when treated with 0.1% (w/v) SDS, and were unable to divide when supplemented with 0.02% SDS. The loss of outer membrane integrity resulted in long-term damage since cell viability was reduced by three orders of magnitude compared to control or uninduced cells. Overexpression of TolAIII did not significantly interfere with the translocation and processing of a plasmid-encoded fusion between the OmpA signal sequence and TEM-beta-lactamase but led to the release of most periplasmic proteins and 90% of the active enzyme into the extracellular fluid. Although the total levels of beta-lactamase accumulation in TolAIII-overproducing cultures was only 1.5- to 2-fold less than in control cells, the formation of periplasmic inclusions bodies was completely suppressed. A threshold concentration of TolAIII was necessary for efficient release of periplasmic proteins since the viability and detergent sensitivity of uninduced cells was comparable to that of control cultures and 80% of the beta-lactamase synthesized remained confined to the periplasm.
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PMID:TolAIII co-overexpression facilitates the recovery of periplasmic recombinant proteins into the growth medium of Escherichia coli. 975 46

Tumor endothelial marker7 (TEM7) is a transmembrane protein that is highly expressed in the tumor endothelium and neurons. In the present study, the expression profile of TEM7 mRNA and protein was investigated in the dorsal root ganglion (DRG) of the rats. In situ hybridization and reverse transcription polymerase chain reaction studies revealed that TEM7 mRNA expression was localized to the neuronal population of the sensory ganglion. Immunohistochemical analysis on TEM protein with specific antibodies further supported the spatial expression patterns of TEM7 mRNA in the DRG. The temporal expression of TEM7 mRNA in the DRG demonstrated a postnatal increase of TEM7 mRNA expression. Our results indicate that TEM7 may play a role in the peripheral sensory neurons of the vertebrate nervous systems.
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PMID:Expression of tumor endothelial marker 7 mRNA and protein in the dorsal root ganglion neurons of the rat. 1670 19

Proteins are key components in a multitude of biological processes, of which the functions carried out by transmembrane (membrane-spanning) proteins are especially demanding for investigations. This is because this class of protein needs to be incorporated into a lipid bilayer representing its native environment, and in addition, many experimental conditions also require a solid support for stabilization and analytical purposes. The solid support substrate may, however, limit the protein functionality due to protein-material interactions and a lack of physical space. We have in this work tailored the pore size and pore ordering of a mesoporous silica thin film to match the native cell-membrane arrangement of the transmembrane protein human aquaporin 4 (hAQP4). Using neutron reflectivity (NR), we provide evidence of how substrate pores host the bulky water-soluble domain of hAQP4, which is shown to extend 7.2 nm into the pores of the substrate. Complementary surface analytical tools, including quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence microscopy, revealed successful protein-containing supported lipid bilayer (pSLB) formation on mesoporous silica substrates, whereas pSLB formation was hampered on nonporous silica. Additionally, electron microscopy (TEM and SEM), light scattering (DLS and stopped-flow), and small-angle X-ray scattering (SAXS) were employed to provide a comprehensive characterization of this novel hybrid organic-inorganic interface, the tailoring of which is likely to be generally applicable to improve the function and stability of a broad range of membrane proteins containing water-soluble domains.
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PMID:Protein-Containing Lipid Bilayers Intercalated with Size-Matched Mesoporous Silica Thin Films. 2807 57