C-terminal proteolytic degradation of recombinant desulfato-hirudin and its mutants in the yeast Saccharomyces cerevisiae

The potent thrombin inhibitor hirudin variant 1, originally isolated from the leech Hirudo medicinalis, was expressed in Saccharomyces cerevisiae under the control of a truncated glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter fragment. Fusion of the yeast acid phosphatase (PHO5) signal sequence to the hirudin gene led to quantitative secretion of recombinant desulfato-hirudin variant 1 (r-hirudin) into the extracellular medium in a growth-dependent manner. In comparison to the genuine molecule, r-hirudin lacks the sulfate group at the Tyr in position 63. Besides the full-length protein of 65 amino acids (hir65), chemical analysis revealed the presence mainly of two derivatives lacking the last amino acid Gln (hir64) or the penultimate Leu (hir63) in addition. When expressing r-hirudin in mutant strains defective in all but one of the three major known carboxypeptidases, it turned out that the vacuolar carboxypeptidase yscY as well as the alpha-factor precursor-processing carboxypeptidase, ysc alpha, participate in the C-terminal degradation of r-hirudin. Direct involvement of yscY and ysc alpha was confirmed by sequential disruption of their structural genes PRC1 and KEX1, respectively. Disruption of PRA1, coding for the yscY-processing proteinase yscA, also abolished yscY-mediated C-terminal r-hirudin degradation, but clearly reduced the overall expression yield. Since ysc alpha is described to be highly specific for basic amino acids which are not present at the C-terminus of r-hirudin, a series of r-hirudin mutants with changes in the C-terminal amino acids were constructed and analysed for ysc alpha-mediated and yscY-mediated degradation. Chromatographic analysis of the expression products confirmed the preference of ysc alpha for basic amino acids, although Tyr, Leu and Gln were also hydrolysed. It could further be concluded that ysc alpha might also be responsible for the C-terminal degradation of recombinant atrial natriuretic factor and epidermal growth factor expressed in yeast.     

Characterization of human immunodeficiency virus type 1 integrase mutants expressed in Escherichia coli

The eight mutant integrase (IN) proteins of human immunodeficiency virus type (HIV-1), which have a single point mutation at a highly conserved central region, were prepared, and characterized in terms of their endonucleolytic activities and disintegration activities in vitro. Mutation of two highly conserved amino acids, Asp116 or Glu152, leads to complete loss of both the activities, suggesting that these two amino acids are directly associated with enzymatic functions. In addition, the mutant of the position Ser147 was found to have highly depressed endonucleolytic activity showing that the reaction was very delayed in comparison with that of the wild type. However, significant disintegration was detected in the mutant Ser147, indicating that the enzymatic mechanisms of the endonucleolytic and disintegration activities are not exactly reverse. The integrase protein with a mutation at the conserved amino acid Asn117 or Gly118 had a slight loss of the endonucleolytic activity, while a mutation at the three positions, Tyr143, Ser153, and Lys159, had no detectable effect on their enzymatic activities. These results indicate that only a few of the conserved amino acids are critical for enzymatic activities.     

Predictable deuteration of recombinant proteins expressed in Escherichia coli

Carrageenan was used to study inflammation-induced changes in spinal nociception and its brain stem modulation in the pentobarbitone-anesthetized rat. Carrageenan was administered intraplantarly into one hindpaw 2 h before the start of electrophysiological single unit recordings of wide-dynamic range (WDR) neurons of the spinal dorsal horn. Carrageenan produced a significant leftward shift in the stimulus-response function for mechanical stimuli, whereas that for noxious heat stimuli was short of statistical significance. Conditioning electrical stimulation in the rostroventromedial medulla (RVM) significantly attenuated noxious heat-evoked, but not mechanically evoked, responses to spinal dorsal horn WDR neurons in the control (contralateral) side. However, in the carrageenan-treated side RVM stimulation had no significant effect on mechanically or noxious heat-evoked responses. Following direct spinal administration of neuropeptide FF (NPFF), noxious heat-evoked responses, but not mechanically evoked responses, were attenuated by RVM-stimulation also in the carrageenan-treated side. This selective NPFF-induced enhancement of brain stem-spinal inhibition was not reversed by naloxone. The results indicate that carrageenan-induced inflammation significantly changes the response properties of spinal nociceptive neurons and their brain stem-spinal modulation. During inflammation, NPFF in the spinal cord produces a submodality-selective potentiation of the antinociceptive effect induced by brain stem-spinal pathways, independent of naloxone-sensitive opioid receptors.     

Mutagenesis of a proton linkage pathway in Escherichia coli manganese superoxide dismutase

Mutagenesis of Escherichia coli manganese superoxide dismutase (MnSD) demonstrates involvement of the strictly conserved gateway tyrosine (Y34) in exogenous ligand interactions. Conservative replacement of this residue by phenylalanine (Y34F) affects the pH sensitivity of the active-site metal ion and perturbs ligand binding, stabilizing a temperature-independent six-coordinate azide complex. Mutant complexes characterized by optical and electron paramagnetic resonance (EPR) spectroscopy are distinct from the corresponding wild-type forms and the anion affinities are altered, consistent with modified basicity of the metal ligands. However, dismutase activity is only slightly reduced by mutagenesis, implying that tyrosine-34 is not essential for catalysis and may function indirectly as a proton donor for turnover, coupled to a protonation cycle of the metal ligands. In vivo substitution of Fe for Mn in the MnSD wild-type and mutant proteins leads to increased affinity for azide and altered active-site properties, shifting the pH-dependent transition of the active site from 9.7 (Mn) to 6.4 (Fe) for wt enzyme. This pH-coupled transition shifts once more to a higher effective pKa for Y34F Fe2-MnSD, allowing the mutant to be catalytically active well into the physiological pH range and decreasing the metal selectivity of the enzyme. Peroxide sensitivities of the Fe complexes are distinct for the wild-type and mutant proteins, indicating a role for Y34 in peroxide interactions. These results provide evidence for a conserved peroxide-protonation linkage pathway in superoxide dismutases, analogous to the proton relay chains of peroxidases, and suggests that the selectivity of Mn and Fe superoxide dismutases is determined by proton coupling with metal ligands.     

Proteolysis of Saccharomyces cerevisiae RNase H1 in E coli

Expression of S cerevisiae RNase H1 in E coli leads to the formation of a proteolytic product with a molecular mass of 30 kDa that is derived from the 39-kDa full length protein. The 30-kDa form retains RNase H1 activity, as determined by renaturation gel assay. The amount of proteolysis observed depends on the procedure used in preparing the cell extracts for protein analysis. The cleavage site on the amino acid sequence of the 39-kDa RNase H1 was determined by N-terminal sequence analysis of the 30-kDa proteolytic form. The cut occurs between two arginines located at the amino terminus region of the protein. The pattern of proteolysis was examined for both the wild-type RNase H1 and a mutant RNase H1 that was constructed in this work. In the mutant the second arginine of the cleavage site was changed to a lysine. Comparisons of the expression of the wild-type and altered protein in two different E coli strains demonstrate that the protease responsible for the degradation has a specificity very similar to that of the OmpT protease. However, the proteolysis observed in an OmpT background in extracts, prepared by boiling the cells in SDS containing buffer, indicates that the protease may, unlike OH108.     

Characterization of recombinant guinea pig alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli. Kinetics, chemical modification and mutagenesis

A recombinant form of guinea pig alkyl-dihydroxyacetonephosphate synthase, a key enzyme in the biosynthesis of ether phospholipids, was characterized. Kinetic analysis yielded evidence that the enzyme operates by a ping-pong rather than a sequential mechanism. Enzyme activity was irreversibly inhibited by N-ethylmaleimide, p-bromophenacylbromide and 2,4-dinitrofluorobenzene. The enzyme could be protected against the inactivation by either of these three compounds by the presence of saturating amounts of the substrate palmitoyl-dihydroxyacetonephosphate. The rate of inactivation of the enzyme by p-bromophenacylbromide was strongly pH dependent and the highest at alkaline conditions. Collectively, these results are indicative of cysteine, histidine and lysine residues, respectively, at or close to the active site. The divalent cations Mg2+, Zn2+ and Mn2+ were found to be inhibitors of enzymatic activity, whereas Ca2+ had no effect. Mutational analysis showed that histidine 617 is an essential amino acid for enzymatic activity: replacement of this residue by alanine resulted in complete loss of enzymatic activity. A recombinant enzyme with the C-terminal five amino acids deleted was shown to be inactive, indicating an important role of the C-terminus for catalytic activity.     

High level, context dependent misincorporation of lysine for arginine in Saccharomyces cerevisiae a1 homeodomain expressed in Escherichia coli

The Saccharomyces cerevisiae a1 homeodomain is expressed as a soluble protein in Escherichia coli when cultured in minimal medium. Nuclear magnetic resonance (NMR) spectra of previously prepared a1 homeodomain samples contained a subset of doubled and broadened resonances. Mass spectroscopic and NMR analysis demonstrates that the heterogeneity is largely due to a lysine misincorporation at the arginine (Arg) 115 site. Arg 115 is coded by the 5'-AGA-3' sequence, which is quite rare in E. coli genes. Lower level mistranslation at three other rare arginine codons also occurs. The percentage of lysine for arginine misincorporation in a1 homeodomain production is dependent on media composition. The dnaY gene, which encodes the rare 5'-AGA-3' tRNA(ARG), was co-expressed in E. coli with the a1-encoding plasmid to produce a homogeneous recombinant a1 homeodomain. Co-expression of the dnaY gene completely blocks mistranslation of arginine to lysine during a1 overexpression in minimal media, and homogeneous protein is produced.     

Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli

The mature hen avidin encoded by a synthetic cDNA was expressed in Escherichia coli in an insoluble form. After resolubilization, renaturation and purification, a recovery of about 20 mg/l cell culture was obtained. ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins. In addition, an acidic avidin mutant, bearing the substitutions Lys3-->Glu, Lys9--> Glu, Arg26-->Asp and Arg124-->Leu of four exposed basic residues, was produced. The protein, expressed and renatured as wild-type avidin, showed unaltered biotin-binding activity. The acidic pI (approximately 5.5) and lack of aggregation of the mutant allowed easy electrophoretic analysis under non-denaturing conditions of the protein alone and of its complexes with biotin, biotinylated transferrin or peroxidase. Analysis of the sera from sensitized subjects revealed that the avidin mutant has altered antigenicity. Both recombinant avidins were crystallized and the three-dimensional structures solved by molecular replacement and refined to 0.22 nm resolution. The three-dimensional structures of the two recombinant molecules, in the absence of biotin and of glycosylation, are fully comparable with those of the natural hen avidin previously reported.     

Growth in iron-enriched medium partially compensates Escherichia coli for the lack of manganese and iron superoxide dismutase

Enrichment of the growth medium with iron partially relieves the phenotypic deficits imposed on Escherichia coli by lack of both manganese and iron superoxide dismutases. Thus iron supplementation increased the aerobic growth rate, decreased the leakage of sulfite, and diminished sensitivity toward paraquat. Iron supplementation increased the activities of several [4Fe-4S]-containing dehydratases, and this was seen even in the presence of 50 microg/ml of rifampicin, an amount which completely inhibited growth. Assessing the O-2 scavenging activity by means of lucigenin luminescence indicated that the iron-enriched sodAsodB cells had gained some means of eliminating O-2, which was not detectable as superoxide dismutase activity in cell extracts. It is noteworthy that iron-enriched cells were not more sensitive toward the lethality of H2O2 despite having the usual amount of catalase activity. This indicates that iron taken into the cells from the medium is not available for Fenton chemistry, but is available for reconstitution of iron-sulfur clusters. We suppose that oxidation of the [4Fe-4S] clusters of dehydratases by O-2 and their subsequent reductive reconstitution provides a mechanism for scavenging O-2 and that speeding this reductive reconstitution by iron enrichment both spared other targets from O-2 attack and maintained adequate levels of these enzymes to meet the metabolic needs of the cells.     

Purification of recombinant human rhinovirus 14 3C protease expressed in Escherichia coli

A physiologically relevant thrombopoietin (TPO) must be a humoral regulator with lineage specificity for megakaryocytes and their precursors. It should be capable of stimulating platelet production in normal animals, and elevated levels of TPO should be detectable in the plasma following acute, severe thrombocytopenia. Acute thrombocytopenia provides a model system that is likely to predict the effects of TPO, since many of the effects on megakaryocytes and platelets observed after induction of acute thrombocytopenia would be mediated by TPO. Important questions remain to be answered. Do the currently available data for the c-Mpl ligand explain previously published data that describe elevated levels of Meg-CSF in the circulation following production of bone marrow aplasia? Does the c-Mpl ligand account for all of the megakaryocyte stimulatory factors that have been described? Is there another factor that accounts for at least some of the acute alterations in megakaryocytopoiesis that occur immediately following a decrease in platelet levels?     

Gene length and codon usage bias in Drosophila melanogaster, Saccharomyces cerevisiae and Escherichia coli

The relationship between gene length and synonymous codon usage bias was investigated in Drosophila melanogaster, Escherichia coli and Saccharomyces cerevisiae. Simulation studies indicate that the correlations observed in the three organisms are unlikely to be due to sampling errors or any potential bias in the methods used to measure codon usage bias. The correlation was significantly positive in E.coli genes, whereas negative correlations were obtained for D. melanogaster and S.cerevisiae genes. When only ribosomal protein genes were used, whose expression levels are assumed to be similar, E.coli and S.cerevisiae showed significantly positive correlations. For the two eukaryotes, the distribution of effective number of codons was different in short genes (300-500 bp) compared with longer genes; this was not observed in E.coli. Both positive and negative correlations can be explained by translational selection. Energetically costly longer genes have higher codon usage bias to maximize translational efficiency. Selection may also be acting to reduce the size of highly expressed proteins, and the effect is particularly pronounced in eukaryotes. The different relationships between codon usage bias and gene length observed in prokaryotes and eukaryotes may be the consequence of these different types of selection.     

Structural and kinetic properties of nonglycosylated recombinant Penicillium amagasakiense glucose oxidase expressed in Escherichia coli

The gene coding for Penicillium amagasakiense glucose oxidase (GOX; beta-D-glucose; oxygen 1-oxidoreductase [EC 1.1.3.4]) has been cloned by PCR amplification with genomic DNA as template with oligonucleotide probes derived from amino acid sequences of N- and C-terminal peptide fragments of the enzyme. Recombinant Escherichia coli expression plasmids have been constructed from the heat-induced pCYTEXP1 expression vector containing the mature GOX coding sequence. When transformed into E. coli TG2, the plasmid directed the synthesis of 0.25 mg of protein in insoluble inclusion bodies per ml of E. coli culture containing more than 60% inactive GOX. Enzyme activity was reconstituted by treatment with 8 M urea and 30 mM dithiothreitol and subsequent 100-fold dilution to a final protein concentration of 0.05 to 0.1 mg ml-1 in a buffer containing reduced glutathione-oxidized glutathione, flavin adenine dinucleotide, and glycerol. Reactivation followed first-order kinetics and was optimal at 10 degrees C. The reactivated recombinant GOX was purified to homogeneity by mild acidification and anion-exchange chromatography. Up to 12 mg of active GOX could be purified from a 1-liter E. coli culture. Circular dichroism demonstrated similar conformations for recombinant and native P. amagasakiense GOXs. The purified enzyme has a specific activity of 968 U mg-1 and exhibits kinetics of glucose oxidation similar to those of, but lower pH and thermal stabilities than, native GOX from P. amagasakiense. In contrast to the native enzyme, recombinant GOX is nonglycosylated and contains a single isoform of pI 4.5. This is the first reported expression of a fully active, nonglycosylated form of a eukaryotic, glycosylated GOX in E. coli.     

Cross-species aminoacylation of tRNA with a long variable arm between Escherichia coli and Saccharomyces cerevisiae

Prokaryotes have three amino acid-specific class II tRNAs that possess a characteristic long variable arm, tRNASer, tRNALeuand tRNATyr, while eukaryotes have only two, tRNASerand tRNALeu. Because of such a phylogenetic divergence in the composition of tRNA, the class II tRNA system is a good candidate for studying how the tRNA recognition manner has evolved in association with the evolution of tRNA. We report here a cross-species aminoacylation study of the class II tRNAs, showing the unilateral aminoacylation specificity between Escherichia coli and a yeast, Saccharomyces cerevisiae. Both SerRS and LeuRS from E.coli were unable to aminoacylate yeast class II tRNAs; in contrast, the yeast counterparts were able to aminoacylate E.coli class II tRNAs. Yeast seryl-tRNA synthetase was able to aminoacylate not only E.coli tRNASerbut also tRNALeuand tRNATyr, and yeast LeuRS was able to aminoacylate not only E.coli tRNALeubut also tRNATyr. These results indicate that the recognition manner of class II tRNA, especially the discrimination strategy of each aminoacyl-tRNA synthetase against noncognate class II tRNAs, is significantly divergent between E.coli and yeast. This difference is thought to be due mainly to the different composition of class II tRNAs in E.coli and yeast.     

Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli

Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BDeltaCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.     

Characterization of human T-cell leukemia virus type I integrase expressed in Escherichia coli

The C-terminal part of the pol gene of the human T-cell leukemia virus type I (HTLV-I) is predicted to encode the integrase (IN) of the virus; however, this protein has not yet been detected in virions or infected cells. We expressed the putative IN from an infectious molecular clone of HTLV-I in Escherichia coli. Comparison with protein resulting from coexpression of HTLV-I protease (PR) and Pol in insect cells indicated that the bacterially expressed protein is identical with or very similar to IN released from a PR-Pol precursor by proteolytic cleavage. HTLV-I IN was purified from E. coli under native conditions. The protein behaved like a dimer in size-exclusion chromatography. It carried out activities characteristic of retroviral IN with high efficiency, displaying a strong preference for U5-derived vs. U3-derived sequences in the processing and strand-transfer reactions. In the disintegration reaction, HTLV-I IN not only accepted the double-stranded branched substrate corresponding to the product of a strand-transfer reaction, but was also able to carry out a phosphoryl transfer on a branched molecule with a single-stranded or a single adenosine overhang.     

Suppressors of superoxide dismutase (SOD1) deficiency in Saccharomyces cerevisiae. Identification of proteins predicted to mediate iron-sulfur cluster assembly

Yeast deficient in the cytosolic copper/zinc superoxide dismutase (SOD1) exhibit metabolic defects indicative of oxidative damage even under non-stress conditions. To help identify the endogenous sources of this oxidative damage, we isolated mutant strains of S. cerevisiae that suppressed metabolic defects associated with loss of SOD1. Six complementation groups were isolated and three of the corresponding genes have been identified. One sod1Delta suppressor represents SSQ1 which encodes a hsp70-type molecular chaperone found in the mitochondria. A second sod1Delta suppressor gene, designated JAC1, represents a new member of the 20-kDa J-protein family of co-chaperones. Jac1p contains a mitochondrial targeting consensus sequence and may serve as the partner for Ssq1p. Homologues of Ssq1p and Jac1p are found in bacteria in close association with genes proposed to be involved in iron-sulfur protein biosynthesis. The third suppressor gene identified was NFS1. Nfs1p is homologous to cysteine desulfurase enzymes that function in iron-sulfur cluster assembly and is also predicted to be mitochondrial. Each of the suppressor mutants identified exhibited diminished rates of respiratory oxygen consumption and was found to have reduced mitochondrial aconitase and succinate dehydrogenase activities. Taken together these results suggest a role for Ssq1p, Jac1p, and Nfs1p in assembly/maturation of mitochondrial iron-sulfur proteins and that one or more of the target Fe/S proteins contribute to oxidative damage in cells lacking copper/zinc SOD.     

A lea-class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae

Although less cytotoxic, the new platinum complex [meso-1,2-bis(2,6-difluoro-4-hydroxyphenyl)-ethylenediamine]sulfatopl atinum (II) (2) is equipotent to cisplatin (1) in the oestrogen-dependent MXT mammary tumour of the mouse. As this may be due to oestrogen level-lowering properties, we compared the effect of 1 and 2 on steroidogenesis in the rat. A single dose of 1 and 2 (20 mumol/kg s.c.) decreased plasma testosterone level in male rats by 90% (1, day 3) and 80% (2, day 7). Luteinizing hormone level remained unchanged in intact and in ovariectomized rats. The activities of the following testicular enzymes were decreased (day 7): cholesterol side-chain cleavage enzyme (1: 33%; 2: 36%), 3 beta-hydroxysteroid dehydrogenase/delta 4,delta 5-isomerase (1: 31%; 2: 48%) and 17 alpha-hydroxylase/17,20-lyase (1: 21%; 2: 15%). Testicular microsomal cytochrome P450 content was also diminished (1: 60%; 2: 49%, day 7). Corticosterone level in plasma and biosynthesis in adrenal explants was not affected, indicating the selectivity of action at the gonadal level. In vitro, neither 1 nor 2 (2 and 20 microM) influenced binding of human chorionic gonadotropin to testis interstitial cells during an observation period up to 21 h. These results suggest that 1 and 2 act at the gonadal level by inhibiting the expression of the steroidogenic enzymes. They do not, however, inactivate the luteinizing hormone receptor.