Growth-associated synthesis of recombinant human glucagon and human growth hormone in high-cell-density cultures of Escherichia coli

Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at high cell concentrations of recombinant Escherichia coli. The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment is presumed invariable during growth and recombinant protein synthesis. Via exponential feeding in the two-phase fed-batch operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant product formation in the recombinant E. coli strains. The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased to 15 g fusion growth hormone 1(-1) and 7 g fusion glucagon 1(-1). The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein during the whole induction period. The stressful conditions of cultivation employed (i.e., high-cell-density cultivation at low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency of synthesized heterologous proteins. The synthesis of the recombinant fusion proteins was strongly growth-dependent and more efficient at a higher specific growth rate. The mechanism linking specific growth rate with recombinant protein productivity is likely to be related to the change in cellular ribosomal content.     

The Legionella micdadei flagellin: expression in Escherichia coli K 12 and DNA sequence of the gene

To study the structure and function of the Legionella flagellum, we screened a genomic L. micdadei library in Escherichia coli for expression of the flagellin (Fla) subunit. One recombinant clone, JM105 (pHI5588), producing a truncated Fla protein of 40.5 kDa was identified. The plasmid pHI5588 carried a L. micdadei DNA insert of 5 kb, containing ca 95% of the fla gene. The complete DNA sequence of the L. micdadei fla gene was obtained by combining sequence data from pHI5588 with results using a polymerase chain reaction-based system for genome walking (vectorette PCR). The L. micdadei fla gene shared a high degree of homology with other flagellin genes in the amino- and carboxy termini, whereas the central region was found to be nonconserved. The fla sequence will facilitate the cloning of Fla proteins from other Legionella species and the study of flagella in the pathogenesis of Legionnaires' disease.     

Influence of incubation conditions on survival and acid tolerance response of Escherichia coli O157:H7 and non-O157:H7 isolates exposed to acetic acid

OBJECTIVE: Recently a few cases of long QT syndrome were reported during treatment with cisapride. In most of these cases, risk factors for cardiac arrhythmias or pharmacologic interactions might have been involved, and the role of cisapride remained unclear. Macrolides such as clarithromycin potentially interact with the metabolic elimination of cisapride and have overlapping indication areas. We therefore studied whether combined treatment with clarithromycin and cisapride leads to pharmacokinetic changes and increased QT intervals. METHODS: The study was an open, randomized, 2-way crossover study with washout periods of 1 week. Twelve healthy volunteers were recruited. Treatments were cisapride (10 mg 4 times a day) for 10 days with concomitant clarithromycin (500 mg twice a day) from days 6 through 10, or clarithromycin (500 mg twice a day) for 10 days combined with cisapride (10 mg 4 times a day) from days 6 through 10. Frequent ECG recordings were performed for 24 hours before drug treatment (baseline). After 5 days of monotherapy and combination therapy, frequent ECG recordings and assessments of plasma drug levels were performed for 24 hours. RESULTS: Clarithromycin alone was associated with a minimal increase in QTc intervals. Monotherapy with 10 mg cisapride 4 times a day led to a concentration-dependent QTc elevation, amounting to 6 ms during steady state. Combination of cisapride and clarithromycin caused an average QTc increase of 25 ms above pretreatment values and 3-fold increases in cisapride concentrations. CONCLUSIONS: QTc elevations after cisapride or clarithromycin alone remained within the normal range of diurnal variation. Coadministration of cisapride and clarithromycin produced a substantial QT prolongation. The data support the recently purported interaction between cisapride and clarithromycin and thus the filed contraindication to combine these drugs.     

Identification of the fucose synthetase gene in the colanic acid gene cluster of Escherichia coli K-12

GDP-L-fucose, the substrate for fucosyltransferases for addition of fucose to polysaccharides or glycoproteins in both procaryotes and eucaryotes, is made from GDP-D-mannose. L-Fucose is a component of bacterial surface antigens, including the extracellular polysaccharide colanic acid produced by most Escherichia coli strains. We previously sequenced the E. coli colanic acid gene cluster and identified one of the GDP-L-fucose biosynthetic pathway genes, gmd. We report here the identification of the gene (fcl), located downstream of gmd, encoding the fucose synthetase.     

In vivo cloning of genes of Escherichia coli K12 by means of homologue recombination

OBJECTIVES: This study was conducted to compare microleakage of two new dentin bonding agents on freshly extracted teeth, cryopreserved teeth, or teeth stored in water containing 0.5% chloramine at 4 degrees C. METHODS: Rectangular Class V cavity preparations were made on the buccal and the lingual surface of wisdom teeth. They were filled with either Scotchbond Multi-Purpose and Z100 (3M Dental Products) or with Gluma 2000 and Pekafill (Bayer Dental). After thermocycling, silver staining penetration was evaluated under a light microscope. SEM examination and EDX analysis were performed to evaluate the microleakage pattern. The results were analyzed by the use of a two-way analysis of variance. RESULTS: Cryopreservation for 13 wk or 12 d refrigeration did not produce changes in the amount of microleakage. However, 48 d or longer of refrigeration increased microleakage. There was no correlation between changes in microleakage and storage time. Specimens prepared with both dentin bonding agents exhibited the same microleakage values and the same microleakage pattern. SIGNIFICANCE: Refrigeration at 4 degrees C in 0.5% chloramine for 48 d or longer may cause an increase in microleakage. Cryopreservation for 13 wk or short-term refrigeration did not affect the microleakage.     

Purification and characterization of the Escherichia coli K1 neuB gene product N-acetylneuraminic acid synthetase

The present studies were designed to evaluate the developmental toxicity of chlorpropham in mice. The first study was conducted to determine administration time, and the second study was designed to evaluate dose-response effects. Chlorpropham was administered to pregnant mice by gavage on Days 8, 8.3, 9, 9.3, 10, and 11 of gestation at a level of 3000 mg/kg bw, and the females were killed on Day 18 of gestation. The administration on Day 8.3 of gestation induced the highest percentage of external malformations with brachyury occurring among more litters than in other groups. Chlorpropham was administered to pregnant mice by gavage at a level of 0 (control), 750, 1500, and 3000 mg/kg bw on Day 8.3 of gestation, and the females were killed on Day 18 of gestation. The total resorption rate was significantly increased in the 3000 mg/kg bw group. The average fetal body weight of each sex was significantly reduced in the 3000 mg/kg treatment group. The total incidence of external malformations was significantly increased in the two highest dose groups in a dose-related manner. Again brachyury was significantly increased in the 3000 mg/kg bw group.     

Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12

Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail. Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined. The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined. Intact cells formed sulfite from sulfonate-sulfur. In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited. In contrast, none of three sulfonates had a competitive effect on sulfate assimilation. In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited. In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization. An E. coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate. In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor. These findings indicate that sulfonate utilization may share some intermediates (e.g., sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization. Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist.     

A mechanism for valine-resistant growth of Escherichia coli K-12 supported by the valine-sensitive acetohydroxy acid synthase IV activity from ilvJ662

Acetohydroxy acid synthase (EC 4.1.3.18; AHAS) isozymes I and III are expressed in Escherichia coli strain K-12 and, when inhibited by L-valine, cannot support cell growth. AHAS IV, expressed from mutation at ilvJ662, exhibits valine-sensitivity similar to that of AHAS III, yet AHAS IV does support cell growth in valine minimal medium. Rate equations were derived for AHAS III and AHAS IV reaction in crude extracts and for partially purified AHAS IV. Values of kinetic constants in these equations were determined in order to model a probable reaction mechanism. Computer modeling of initial velocity reactions at physiological substrate concentrations simulated consequences of valine-inhibition and revealed that AHAS IV synthesized AHB at a maximal rate over four times faster than AHAS III under these conditions. The simulations predicted that cells depending upon AHAS III for growth in valine minimal medium would accumulate higher levels of 2-ketobutyrate than cells using AHAS IV. Experiments on growth inhibition by valine revealed more than a five-fold difference in 2-ketobutyrate accumulation, thus confirming these predictions. These data support the hypothesis that valine inhibition of growth is a consequence of 2-ketobutyrate accumulation to toxic levels. We propose that the valine-inhibited AHAS IV activity prevents growth inhibition by keeping 2-ketobutyrate accumulation to a lower level than resulting from AHAS III activity.     

Metabolites influence control of lysine transfer ribonucleic acid synthetase formation in Escherichia coli K-12

A mutant of E. coli K-12 has been isolated which has only 1-3% of the wild-type lysyl-tRNA synthetase activity [L-lysine:tRNA ligase (AMP forming), EC 6.1.1.6]. Additions of 20 mM L-alanine or 6 mM leucine dipeptides to the culture medium can restore the activity of lysyl-tRNA synthetase in the mutant strain to the wild-type level. Experiments on the in vivo charging of lysine tRNA in the mutant show that in the absence of the metabolites lysine tRNA is charged 15-23%. Upon the addition of 3 mM L-leucyl-L-alanine to the medium the lysyl tRNA synthetase activity increases 25-fold and the in vivo charging of lysine tRNA returns to the wild-type level. Experiments with antibody against lysyl-tRNA synthetase show that the stimulation of lysyl-tRNA synthetase activity by the metabolites is the result of new protein synthesis.     

Determination of ionic species formed during growth of Escherichia coli by capillary isotachophoresis

The ionic species that are formed during the microbial growth of Escherichia coli were determined by capillary isotachophoresis as a function of the time of cultivation. This formation was indicated by the change in a sum parameter, the impedance of the nutrient broth, measured by a special electrode system. Based on the determination of the individual ions formed under the given conditions (identified as acetate, lactate, alpha-ketoglutarate, fumarate, ammonium and probably a simple amine), the change in conductivity was calculated and compared with that obtained by the impedance measurement of the bulk medium. From the results it can be concluded that the change in the sum parameter as a function of time is originated by the ions determined.     

Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli

A new variant of human growth hormone was recently found [Pavlu, B. & Gellerfors, P. (1993) Bioseparation 3, 257-265]. We report here the identification and the structural determination of this variant. The variant, which is formed during the expression of human growth hormone in Escherichia coli, was found to be more hydrophobic than rhGH as judged by its prolonged elution time by hydrophobic interaction chromatography. The rhGH hydrophobic variant (rhGH-HV) was isolated and subjected to trypsin digestion and RP-HPLC analysis, resulting in an altered retention time of one single tryptic peptide as compared to the corresponding fragment of rhGH. This tryptic peptide constitutes the C-terminus (aa 179-191) of hGH and contains one of the two disulfide bridges in hGH, viz. Cys182-Cys189. Amino acid sequences and composition analyses of the tryptic peptide from rhGH-HV (Tv18-19) and the corresponding tryptic peptide from rhGH (T18+19) were identical. Electrospray mass spectrometry (ES MS) of Tv18+19 isolated from rhGH-HV revealed a monoisotopic mass increase of 32.7, as compared to T18+19 from rhGH. A synthetic Tv18+19 peptide having a trisulfide bridge between Cys182 and Cys189 showed identical fragment in ES/MS compared to Tv18+19 isolated from rhGH-HV, i.e. m/z 617.7 and 682.9. These fragments are formed through a unique cleavage in the trisulfide (Cys182-SSS-Cys189) bridge not found in the corresponding T18+19 disulfide peptide. Furthermore, the synthetic Tv18+19 co-eluted in RP-HPLC with Tv18+19 isolated from rhGH-HV. Two-dimensional NMR spectroscopy of the synthetic T18+19 and Tv18+19 peptides were performed. Using these data all protons were assigned. The major chemical shift changes (delta delta > 0.05 ppm) observed were for the beta-protons of Cys182 and Cys189 in Tv18+19 as compared to T18+19. CD spectroscopy data were also in agreement with the above results. Based on these physico-chemical data rhGH-HV has been structurally defined as a trisulfide variant of rhGH. The receptor binding properties of rhGH-HV was studied by a biosensor device, BIAcore. The binding capacity of rhGH-HV was similar to rhGH with a binding stoichiometry to the rhGHBP of 1:1.6 and 1:1.5, respectively, indicating that the trisulfide modification did not affect its receptor binding properties.     

Differences in the cellular surface of hybrid Escherichia coli K12 bacteria inheriting the rfb a3,4 gene of Shigella flexneri detected using atomic-force microscopy

The cell surface of E. coli initial parent strain K12 J62 his-, chemotype Ra, and E. coli transductant strain K12 J62 his+, acquiring the capacity for synthesizing primary S-specific side chains of the lipopolysaccharide of S. flexneri O-antigen (group-specific factor 3,4), was studied by the method of atomic force microscopy. The comparative analysis of the images of the genetically linked pair of E.coli strains K12 J62 revealed the presence of essential differences in the topography of the surface structure of the compared bacterial cells, differing in their capacity for synthesizing S. flexneri factor 3,4 represented by repeating chains of L-rhamnose and N-acetyl-D-glucosamine.     

Overproduction of a functional fatty acid biosynthetic enzyme blocks fatty acid synthesis in Escherichia coli

beta-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase.     

Glucose transport as rate-limiting step in the growth of Escherichia coli on glucose

Over a wide range of growth rates, two strains of Escherichia coli growing aerobically in continuous culture under glucose limitation utilized glucose at rates identical with those at which cells harvested from the chemostats transported [14C]glucose.     

Biochemical characterization of a delta12 acyl-lipid desaturase after overexpression of the enzyme in Escherichia coli

OBJECTIVE: To study the variability in urine composition with respect to factors of importance for the calcium salt crystallization process and to test the reliability of using one or several urine samples in the clinical evaluation. PATIENTS AND METHODS: Twelve patients collected 16-hour daytime and 8-hour night urine samples during 4 days of the same week. The urine was analysed for calcium, oxalate, phosphate, magnesium, citrate and pH, and the ion activity products of CaOx [AP(CaOx) index] and CaP were calculated. The risk of CaOx crystallization, as well as the inhibition of CaOx crystal growth and aggregation, were assessed. RESULTS: There was a good correlation between estimates of the AP(CaOx) index in the different samples, as well as between the AP(CaOx) index and the direct assessment of the risk of CaOx crystallization in the night and daytime urine samples. There was, however, a pronounced intra-individual variation of all variables and parameters. With the assumption that an abnormality would appear in at least one of the four samples, we found that in more than 80% of the cases, two 24-hour (16 + 8 h) urine samples were sufficient to establish whether the patient had a normal or an abnormal urine composition. CONCLUSION: Urine samples collected during two 24-, 16- or 8-hour periods appear to be useful for detecting biochemical abnormalities considered of importance for CaOx stone formation.