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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The processing of LamB-IGF-1 fusion protein and the export of processed IGF-1 (insulin-like growth-factor-1) into the growth medium was examined in the Escherichia coli host strain, JM101. Several strain or plasmid modifications were tried to increase export of periplasmic (processed) IGF-1 into the growth medium of JM101. These included: (1) use of a lon null mutant strain to increase accumulation levels of unprocessed LamB-IGF-1 fusion protein; (2) use of an alternative drug resistance marker on the expression plasmid rather than beta-lactamase, thereby reducing any competition for processing of LamB-IGF-1 by signal peptidase; (3) examination of whether phage M13 gene III protein expression caused more periplasmic IGF-1 to be exported into the growth medium due to increased outer membrane permeability; and (4) examination of the effect of E. coli or yeast optimized IGF-1 codons. None of these strain or plasmid modifications caused any significant increase in export of IGF-1 into the growth medium of JM101. Solubility studies of LamB-IGF-1 and processed IGF-1 showed that virtually all of the LamB-IGF-1 and IGF-1 remaining within the cell after a 2 h induction period was insoluble. This implied that only soluble LamB-IGF-1 was processed to IGF-1 and that only soluble IGF-1 was exported into the growth medium. Taken together, the results indicated that LamB-IGF-1 and IGF-1 solubility were the limiting factors in secretion of IGF-1 into the periplasm and export of IGF-1 into the growth medium.
Mol Gen Genet 1988 Dec
PMID:Secretion and export of IGF-1 in Escherichia coli strain JM101. 307 40

Investigation of possible variations between prokaryotic and eukaryotic signal sequences of exported proteins has revealed unexpected differences. Apart from the known similarities (presence of a core hydrophobic sequence preceded by a positively charged amino terminus and followed by a flexible structure), we have found that the core is much more rigid in eukaryotic signals than in their prokaryotic counterparts, and that at both ends the constraints are much more stringent in bacteria than in human cells. The differences have been summarized as a set of 17 criteria describing noteworthy features discriminating between the two classes of signal peptides. The program we used permitted each class of sequences to be learned; Escherichia coli sequences were well learned (i.e., they could be recognized by the programs as having common features), whereas human sequences were found to exhibit a much wider variation. Thus it was possible to propose a consensus in the case of the bacterial peptides, but none (or a much looser one) in the case of the human sequences. Two sequences were exceptional among the E. coli signal peptides, those of lipoprotein and plasmid-borne beta-lactamase, suggesting that they have special origins or destinations. Finally, the differences found strongly suggest that the mode of secretion is rather different in the two types of organisms, in spite of the common features of the signal sequences.
J Mol Evol 1986
PMID:Protein export in prokaryotes and eukaryotes: indications of a difference in the mechanism of exportation. 310 13

The endo-beta-1,3-1,4-glucanase enzyme of Bacillus subtilis C120, when synthesized in Escherichia coli, is located mainly in the cytoplasm, but enzyme activity is also detected in the periplasmic space and in the extracellular medium. The proportion recovered in the extracellular medium is not altered by changes in the levels of synthesis of the enzyme. Lysis of E. coli cells is ruled out as the cause of the secretion by the normal localization of beta-galactosidase, an intracellular protein. However, beta-lactamase, which is normally found in the periplasmic space, is detected in the extracellular medium of E. coli transformants containing beta-glucanase plasmids, suggesting that the presence of beta-glucanase in the cell alters the permeability of the outer membrane. The beta-glucanase proteins found in the extracellular medium, the periplasmic space and the cytoplasm have the same electrophoretic mobilities as the secreted enzyme of B. subtilis. Amino-terminal sequencing has shown that the beta-glucanase enzyme in the intracellular fraction of E. coli is processed at a site two amino acids distant from the processing site used in B. subtilis.
Mol Microbiol 1988 Nov
PMID:Secretion and processing of the Bacillus subtilis endo-beta-1,3-1,4-glucanase in Escherichia coli. 314 87

Utilizing an improved method for colony hybridization developed by Haas & Fleming, biotin and 32P-labelled TEM-1 probes were compared for sensitivity and specificity in identifying the type of beta-lactamase made by over 100 clinical bacterial isolates. The new procedure was more reliable than a standard one, but still gave more than 20% false positive and false negative reactions.
Mol Cell Probes 1988 Mar
PMID:Reliability of biotinylated DNA probes in colony hybridization: evaluation of an improved colony lysis method for detection of TEM-1 beta-lactamase. 326 6

The Ser68(AGC) codon of the beta-lactamase gene was changed to the glycine codons GGA and GGC. With glycine at position 68, beta-lactamase is inactive because it does not have a nucleophilic side-chain to function in the reaction mechanism. The mutant SG68(GGA) allele had no detectable beta-lactamase activity; however, the mutant SG68(GGC) did produce a small amount of activity. Both mutant alleles produce comparable amounts of beta-lactamase protein in a maxi-cell system. To identify why these two "same-sense" beta-lactamase mutants differ phenotypically, we introduced the alleles into Escherichia coli strains with mutations that affect translational fidelity. The rpsD mutation, which decreases fidelity, significantly increased activity with the SG68(GGC) allele, while the rpsL mutation, which increases translational fidelity, had little effect on the beta-lactamase activity. The rpsD and rpsL alleles had no effect on the SG68(GGA) allele. From the allele specificity of the activity produced by the bla mutants, and from the differential effect of translational fidelity on the activity of the SG68(GGC) allele, we infer that tRNA(GCU)Ser, the AGU/C reading tRNA(Ser), mistranslates SG68(GGC) at a frequency of about 0.1%, and subsequently produces active beta-lactamase. This is the first observation of an A/G wobble with a wild-type tRNA at the first position of the codon-anticodon interaction.
J Mol Biol 1988 May 20
PMID:Evidence for a unique first position codon-anticodon mismatch in vivo. 326 66

To investigate structural characteristics important for eukaryotic signal peptide function in vivo, a hybrid gene with interchangeable signal peptides was cloned into yeast. The hybrid gene encoded nine residues from the amino terminus of the major Escherichia coli lipoprotein, attached to the amino terminus of the entire mature E. coli beta-lactamase sequence. To this sequence were attached sequences encoding the nonmutant E. coli lipoprotein signal peptide, or lipoprotein signal peptide mutants lacking an amino-terminal cationic charge, with shortened hydrophobic core, with altered potential helicity, or with an altered signal-peptide cleavage site. These signal-peptide mutants exhibited altered processing and secretion in E. coli. Using the GAL10 promoter, production of all hybrid proteins was induced to constitute 4-5% of the total yeast protein. Hybrid proteins with mutant signal peptides that show altered processing and secretion in E. coli, were processed and translocated to a similar degree as the non-mutant hybrid protein in yeast (approximately 36% of the total hybrid protein). Both non-mutant and mutant signal peptides appeared to be removed at the same unique site between cysteine 21 and serine 22, one residue from the E. coli signal peptidase II processing site. The mature lipo-beta-lactamase was translocated across the cytoplasmic membrane into the yeast periplasm. Thus the protein secretion apparatus in yeast recognizes the lipoprotein signal sequence in vivo but displays a specificity towards altered signal sequences which differs from that of E. coli.
Mol Microbiol 1988 Mar
PMID:Defective Escherichia coli signal peptides function in yeast. 328 32

Mutants of Escherichia coli K12 carrying exc mutations inducing the release of the plasmid pBR322-encoded beta-lactamase (EC 3.5.2.6) into the extracellular medium were analysed and compared with previously described excretory mutants carrying lky mutations associated with the release of alkaline phosphatase and to tolA and tolB mutants, originally described as tolerant towards various colicins. The exc, lky and tol mutations mapped near the gal operon at min 16.5 of the E. coli linkage map. A genetic analysis presented in this paper showed that some exc and lky mutations belonged to the tolA and tolB complementation groups. Furthermore, we identified a third cistron, excC, involved in the excretion of periplasmic enzymes but distinct from the two others.
Mol Gen Genet 1987 Sep
PMID:tolA, tolB and excC, three cistrons involved in the control of pleiotropic release of periplasmic proteins by Escherichia coli K12. 331 62

A promoter-probe system, based on the ampC beta-lactamase gene of Escherichia coli, has been developed for the isolation and characterization of transcriptional signals in the gram-positive bacterium Streptomyces lividans. The promoter-probe vector, denoted pJAS14, has the SLP1.2 replicon with a copy number of four-five plasmids per cell. It contains a unique BamHI site just in front of the ampC ribosome-binding site, and upstream of this BamHI site a transcriptional terminator signal that prevents readthrough transcription from plasmid-borne promoters has been inserted. Using pJAS14, we have shot-gun cloned chromosomal DNA from S. lividans and S. lavendulae into the BamHI site, and isolated a number of promoter containing DNA fragments by the use of the chromogenic cephalosporin nitrocefin. On plates, we identified promoters of varying strengths and also with differences in nutritional and temporal expression. Using liquid cultures of S. lividans, it has been demonstrated that one promoter, denoted P1 (SEP8), as well as the ampC gene of E. coli, show activity corresponding to the vegetative growth of the cells. The P1 (SEP8) promoter was shown to be expressed also in E. coli, and it initiates RNA synthesis at exactly the same nucleotides in both S. lividans and E. coli. The promoter shows good homology to the E. coli promoter consensus sequence in both the -35 and -10 regions. Thus, this promoter is a representative of the SEP (Streptomyces E. coli-type promoter) class of promoters recently described (Jaurin and Cohen 1985). This indicates that an S. lividans RNA polymerase recognizes the same sequence determinants and chooses the point of initiation of RNA synthesis in the same way as the corresponding E. coli enzyme.
Mol Gen Genet 1987 Nov
PMID:Chromogenic identification of promoters in Streptomyces lividans by using an ampC beta-lactamase promoter-probe vector. 332 38

The coding region for the mature form of TEM beta-lactamase was fused to random positions within the coding region of the penicillin-binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in-frame fusions were determined. All fusion proteins that contained at least the NH2-terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the beta-lactamase moiety had been translocated to the periplasm. Fusion proteins that contained less than or equal to 63 residues of PBP 1B possessed beta-lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the beta-lactamase moiety of these fusion proteins remained in the cytoplasm. The beta-lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic transmembrane segment (residues 64-87), with a short NH2-terminal domain (residues 1-63), and the remainder of the polypeptide (residues 88-844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane.
Mol Microbiol 1987 Jul
PMID:Use of a beta-lactamase fusion vector to investigate the organization of penicillin-binding protein 1B in the cytoplasmic membrane of Escherichia coli. 333 Jul 53

In Gram-negative bacteria, beta-lactam antibiotics must overcome two barriers, the outer membrane and the periplasmic beta-lactamase, before they reach the targets of their action, penicillin-binding proteins. Although the barrier property of the outer membrane and catalytic property of the beta-lactamases have been studied and their significance in creating beta-lactam resistance emphasized, the interaction between these two barriers has not been treated quantitatively. Such treatment shows that the sensitivity, to a variety of beta-lactams, of the Escherichia coli K-12 cells containing very different levels of chromosomally coded AmpC beta-lactamase, or a plasmid-coded TEM-type beta-lactamase, can be predicted rather accurately from the penetration rate through the outer membrane and the hydrolysis rate in the periplasm. We further propose a new parameter, 'target access index', which is a quantitative expression of the result of interaction between the two barriers, and reflects the probability of success for the antibiotic to reach the targets.
Mol Microbiol 1987 Jul
PMID:Sensitivity of Escherichia coli to various beta-lactams is determined by the interplay of outer membrane permeability and degradation by periplasmic beta-lactamases: a quantitative predictive treatment. 333 Jul 55


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