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.     

Effect of growth environment on the stability of a recombinant shuttle plasmid, pCPPS-31, in Escherichia coli

The effect of environmental growth conditions on the segregational stability of a recombinant Escherichia coli-Bacillus subtilis shuttle plasmid pCPPS-31 which expresses carboxymethyl cellulase (CMCase) and contains the neomycin resistance gene (Ner), was studied in E. coli DH5 alpha. The stability increased with the decrease in medium complexity. Lowering culture temperatures had a negative effect on stability. Stability was maximum at 100 rpm and a medium/flask volume ratio of 1:10. A pH range of 5-8 had no significant effect on stability. The recombination (rec) and gyrase (gyr) backgrounds of E. coli hosts had no apparent effect on plasmid stability. The plasmid was structurally stable under all circumstances.     

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?     

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.     

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a K(m) = 0.70 microM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing.     

Incorporation of norleucine at methionine positions in recombinant human macrophage colony stimulating factor (M-CSF, 4-153) expressed in Escherichia coli: structural analysis

Expression of the 17.5-kDa truncated form of human recombinant macrophage colony stimulating factor (rM-CSF, 4-153) in Escherichia coli is complicated by the replacement of methionine residues by norleucine. In order to detect and quantitate this mistranslational event, the intact and the S-carboxyamidomethylated proteins were analyzed by amino acid analysis, automated Edman amino acid sequencing, and electrospray mass spectrometry. In addition, the endoproteinase Glu-C generated peptides were subjected to amino acid sequencing, high-performance liquid chromatography, and electrospray ionization mass spectrometry. The extent of norleucine substitution in different batches of rM-CSF varied between 0% and 20%. The relative instability of methionine residues needs to be considered when calculating the extent of norleucine substitution at methionine positions. The mass spectrometry of the intact rM-CSF allowed for examination of the distribution of multiply substituted methionine to norleucine species, and it enabled detection and quantitation of the norleucine incorporation down to the approximately 3% level. Selective ion chromatograms of molecular ions of interest obtained in reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry of proteolytic fragments offered a reliable and fast method of detection and quantitation of norleucine-containing peptides. Norleucine residues were uniformly distributed among all four methionine positions (10, 27, 61, and 65). A substitution of methionine by its structural norleucine analog does not have any effect on the activity of the refolded rM-CSF dimers.     

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.     

SurA assists the folding of Escherichia coli outer membrane proteins

Many proteins require enzymatic assistance in order to achieve a functional conformation. One rate-limiting step in protein folding is the cis-trans isomerization of prolyl residues, a reaction catalyzed by prolyl isomerases. SurA, a periplasmic protein of Escherichia coli, has sequence similarity with the prolyl isomerase parvulin. We tested whether SurA was involved in folding periplasmic and outer membrane proteins by using trypsin sensitivity as an assay for protein conformation. We determined that the efficient folding of three outer membrane proteins (OmpA, OmpF, and LamB) requires SurA in vivo, while the folding of four periplasmic proteins was independent of SurA. We conclude that SurA assists in the folding of certain secreted proteins.     

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.     

Purification and characterization of D-beta-hydroxybutyrate dehydrogenase expressed in Escherichia coli

D-beta-Hydroxybutyrate dehydrogenase (BDH), a lipid-requiring enzyme, has been cloned into pUC18, expressed in Escherichia coli, and purified to homogeneity. The apoenzyme, i.e., the enzyme devoid of phospholipid, has no activity, but can be activated by phospholipid to a specific activity of 129 mumol/(min.mg). The functional properties of the enzyme expressed in E. coli were compared with the enzyme purified from rat liver. The specific activities, kinetic parameters, and phospholipid activation profiles were virtually identical. These results indicate that the expression of the enzyme in E. coli is a viable method for producing active functional BDH and should allow for the production of specifically altered BDH molecules.     

Gram-scale synthesis of recombinant chitooligosaccharides in Escherichia coli

Basilar membrane (BM) noise, measured as a velocity signal under the quiet acoustic condition, was investigated in the guinea pig. The cochleas of anesthetized young healthy guinea pigs were surgically exposed and a hole was made on the lateral wall of the scala tympani of the first cochlear turn for visualization of the BM and measurement of the BM velocity with a laser interferometer. The amplitude and frequency of the BM velocity noise were analyzed by a spectrum analyzer under different conditions. The spectrum of the BM velocity noise was a band limited function with a peak velocity at the topographic best frequency of the measured location on the BM. The peak velocity ranged to about 8 microm/s and depended on the physiological condition of the cochlea. Saline blockage of the external auditory canal or the middle ear did not change the BM noise. BM noise was much smaller, or was not evident, when the cochlear sensitivity decreased. The suppression tuning curve of the BM velocity noise indicates that the maximum suppression caused by an acoustic pure tone occurred at the best frequency location. A low sound level wide band acoustic noise given to the external ear canal produced a spectrum function having the same frequency and amplitude response as the BM noise. Electrical stimulation of the crossed olivocochlear bundle significantly depresses the BM velocity noise. These data demonstrate that the BM noise is a representation of internal rather than external noise. The amplitude and frequency of the BM noise reflect the usual cochlear sensitivity and frequency selectivity. Since the organ of Corti in the sensitive cochlea is a highly sensitive and tuned mechanical system, the internal (to the animal) noise responsible for the BM noise may originate from mechanical vibrations remote from the cochlea and propagated to the ear, or may be caused by Brownian motion of cellular structures in the cochlea.     

Purification and crystallization of the CDK-associated protein phosphatase KAP expressed in Escherichia coli

The kinase associated phosphatase (KAP) is a human dual specificity protein phosphatase that dephosphorylates the cell cycle control protein, cyclin dependent kinase-2 on Thr 160 in a cyclin dependent manner (Poon & Hunter, 1995). We report here the over-expression of KAP in Escherichia coli as an N-terminal His-tagged protein using a modified pET-28a T7-expression vector. The recombinant protein was purified to homogeneity and crystallized. The crystals diffract to 2.3 A resolution when exposed to synchrotron radiation and belong to space group P6(1)22, or its enantiomorph P6(5)22, with unit cell dimensions a = b = 74.5 A, c = 139.5 A.     

Purification of recombinant apolipoprotein A-1Milano expressed in Escherichia coli using aqueous two-phase extraction followed by temperature-induced phase separation

Immunohistochemistry using anti-human neuron-specific enolase (NSE) mouse monoclonal antibody was performed in human brains from autopsy cases, which enabled us to assess the neuronal damage besides hematoxylin and eosin or Klüver-Barrera stain. Neurons in cerebral neocortex which showed necrotic changes such as prominent cytoplasmic vacuolization or cellular shrinkage with nuclear pyknosis showed a tendency to be less stained by anti-NSE antibody. Anti-NSE immunostaining was statistically significantly less in the neocortex from CO intoxication than from other causes of death, although morphological necrotic changes were less observed in CO intoxication. Hippocampal CA1 neurons clearly lost NSE immunoreactivity with the progression of necrotic changes. Neurons in CA2 were statistically significantly better stained by anti-NSE antibody than in CA1, 3, and 4. Cerebellar Purkinje cells were poorly stained by anti-NSE antibody, whereas neurons in cerebellar dentate nucleus and inferior olive in medulla oblongata were better stained. Anti-NSE immunostaining was lost in the injured areas of the cerebral neocortex while neurons in the intact areas were better stained in brain injury. These results indicate that anti-NSE immunostaining of neurons could reflect vital reaction and could be useful in evaluating neuronal damage in the hippocampal CA1 region or brain injury.     

Characterization of recombinant Saccharomyces cerevisiae manganese-containing superoxide dismutase and its H30A and K170R mutants expressed in Escherichia coli

All known Mn-containing superoxide dismutases (MnSODs) have a highly conserved histidine (His-30 in Escherichia coli FeSOD) in the active-site channel, and nearly all have an active-site arginine (Arg-170) that has been proposed to play a combined structural and functional role [Chan et al., Arch. Biochem. Biophys. 279, 195-201 (1990)]. In Saccharomyces cerevisiae MnSOD, the active-site arginine is replaced by a lysine. The S. cerevisiae MnSOD gene has been cloned and expressed in E. coli, and H30A and K170R site-specific mutants have been prepared. The purified recombinant native (RN) and mutant enzymes were compared to one another and to the native enzyme purified from S. cerevisiae (SC) in terms of activity, temperature stability, and sensitivity to 2,4,6-trinitrobenzenesulfonate (TNBS) and phenylglyoxal (PG). All enzymes had high specific activities (SC = 5000, RN = 5600, H30A = 4500, K170R = 4600) (U/mg, using the pyrogallol assay). SC, RN, and H30A were very stable at 75 degreesC (pH 8.0), with half-lives of 4.7, 2.8, and 2.7 h, respectively, while K170R had a much greater temperature lability, with a half-life of 0.36 h under these conditions. TNBS (0.5 mM, pH 9.0, 25 degreesC) rapidly inactivated SC, RN, and H30A, with half-lives of 3. 5, 5.1, and 5.5 min, respectively, but only slowly inactivated K170R, with a half-life of 101 min. PG (20 mM, pH 9.0, 25 degreesC) caused very slow inactivation of SC, RN, and H30A by biphasic kinetics, and each enzyme retained >/=25% activity after 3 h of modification. K170R, on the other hand, was completely inactivated by PG under these conditions by first-order kinetics, with a half-life of 7.0 min. The data suggest that His-30, a residue highly conserved in the active-site channel of MnSODs and FeSODs, does not play a crucial role in catalysis or stability. In addition, Lys-170, a residue that is almost always arginine in the numerous other MnSODs and FeSODs sequenced to date, can be replaced by arginine with no loss of catalytic activity, but K170R is less stable and Arg-170 in this mutant is more exposed than the corresponding arginine in other SODs. RN and SC showed some surprising differences. Thus, while the specific activities of RN and SC are very similar, SC is more stable to inactivation at 75 degreesC, and less susceptible to inactivation by phenylglyoxal, than RN. These data suggest that there may be slight differences in the tertiary structures of SC, the native enzyme expressed in S. cerevisiae, and RN, the recombinant native enzyme expressed in E. coli.     

Cobalamin-dependent methionine synthase from Escherichia coli: involvement of zinc in homocysteine activation

Methionine synthase (MetH) is a modular protein with at least four distinct regions; amino acids 2-353 comprise a region responsible for binding and activation of homocysteine, amino acids 345-649 are thought to be involved in the binding and activation of methyltetrahydrofolate, amino acids 650-896 are responsible for binding of the prosthetic group methylcobalamin, and amino acids 897-1227 are involved in binding adensylmethionine and are required for reductive activation of enzyme in the cob(II)alamin form. Previous studies have shown that mutations of Cys310 or Cys311 to either alanine or serine result in loss of all detectable catalytic activity. These mutant proteins retain the ability to catalyze methyl transfer from methyltetrahydrofolate to exogenous cob(I)alamin, but have lost the ability to transfer methyl groups from exogenous methylcobalamin to homocysteine [Goulding, C. W., Postigo, D., and Matthews, R. G. (1997) Biochemistry 36, 8082-8091]. We now demonstrate that both MetH holoenzyme and a truncated MetH(2-649) protein, which lacks a cobalamin prosthetic group, contain 0.9 equiv of zinc, while the Cys310Ser and Cys311Ser mutant proteins contain less than 0.05 equiv of zinc. Addition of l-homocysteine to MetH(2-649) is accompanied by release of 1 equiv of protons/mol of protein, while addition of l-homocysteine to the Cys310Ser and Cys311Ser mutant truncated proteins does not result in proton release. The Cys310Ala and Cys311Ala mutant methylcobalamin holoenzymes have completely lost the ability to transfer the methyl group from methylcobalamin to homocysteine, suggesting that zinc is required for this reaction. Further evidence that zinc is required for catalytic activity comes from experiments in which the zinc is removed from MetH(2-1227). Removal of zinc from methylated wild-type holoenzyme by treatment with methyl methanethiolsulfonate and then with dithiothreitol and EDTA results in loss of the ability of the protein to catalyze methyl transfer from methyltetrahydrofolate to homocysteine. Reconstitution of the zinc-depleted holoenzyme results in incorporation of 0.4 equiv of zinc/mol of protein and partial restoration of the ability of the protein to catalyze homocysteine methylation.     

Affinity-assisted in vivo folding of a secreted human peptide hormone in Escherichia coli

We show that coexpression of a specific binding protein in Escherichia coli can significantly improve the relative yields of correctly folded human insulin-like growth factor I (IGF-I). A glutathione redox buffer was used during growth to allow formation and breakage of disulfide bonds in the periplasm of the bacterial host. Both the binding protein and the peptide hormone were produced as affinity fusions, which allowed purification of the in vivo formed heterodimer by alternative affinity purification methods. The use of affinity-assisted in vivo folding has general implications for expression, folding, and purification of recombinant proteins.     

Purification and characteristics of recombinant mouse metallothionein-I from Escherichia coli

A method for simultaneously detecting membrane permeability (characteristic of necrosis) and DNA fragmentation (characteristic of apoptosis) is described. By combining a common dye-exclusion method (Trypan Blue) with a commercially available terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) labeling kit, we have succeeded in developing a novel methodology for obtaining permanently mounted slides of monolayer cell cultures double-labeled for DNA fragmentation and cell lysis. This method should facilitate in situ studies of cell death by allowing for a more accurate quantification of total toxicity in monolayer cell cultures and perhaps further enhance our understanding of the different mechanisms of cell death as well.     

Guanidine-induced denaturation of beta-glycosidase from Sulfolobus solfataricus expressed in Escherichia coli

Guanidine-induced denaturation of Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli, Sbetagly, was investigated at pH 6.5 and 25 degreesC by means of circular dichroism and fluorescence measurements. The process proved reversible when the protein concentration was lower than 0.01 mg mL-1. Moreover, the transition curves determined by fluorescence did not coincide with those determined by circular dichroism, and the GuHCl concentration corresponding at half-completion of the transition increased on raising the protein concentration in the range 0.001-0.1 mg mL-1. Gel filtration chromatography experiments showed that, in the range 2-4 M GuHCl, there was an equilibrium among tetrameric, dimeric, and monomeric species. These findings, unequivocally, indicated that the guanidine-induced denaturation of Sbetagly was not a two-state transition with concomitant unfolding and dissociation of the four subunits. A mechanism involving a dimeric intermediate species was proposed and was able to fit the experimental fluorescence intensity transition profiles, allowing the estimation of the total denaturation Gibbs energy change at 25 degreesC and pH 6.5. This figure, when normalized for the number of residues, showed that, at room temperature, Sbetagly has a stability similar to that of mesophilic proteins.     

Coupling protein stability and protein function in Escherichia coli CspA

Idiopathic thrombocytopenic purpura (ITP, also known as immune thrombocytopenic purpura) in adults is principally a disease of young women. Although in some patients the onset is acute and complete resolution occurs, in most patients, the onset is insidious and the course is chronic. In spite of the relative frequency of ITP, there are important unresolved issues in its diagnosis and management. For this reason, the American Society of Hematology (ASH) chose ITP as the disease topic for its initial sponsored practice guideline in 1993. A major conclusion of the published guideline was the lack of firm evidence on which to base diagnostic procedures and management strategies. This review describes the clinical features of ITP in adults, emphasizes the principal unresolved issues in diagnosis and management, and outlines the critical areas for future research.     

Diagnostic potential of Toscana virus N protein expressed in Escherichia coli

The nucleocapsid (N) protein of the Toscana (TOS) virus was expressed in Escherichia coli by using a pET15b vector. The recombinant protein was purified by affinity chromatography and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The recombinant antigen was reactive with positive human sera, and the reactivity correlated very well (r = 0.9) with that of a whole-virus antigen when tested by EIA with 30 TOS virus-positive and 30 TOS virus-negative serum samples. The results demonstrate that the recombinant N protein can be easily produced in a procaryotic system and used for diagnostic assays for TOS virus immunity.