Biological activity of rat proinsulin synthesized by Escherichia coli

Fused proteins possessing insulin antigenic determinants are synthesized and secreted by several strains of Escherichia coli K-12 bearing plasmids containing cDNA copies of rat proinsulin mRNA. Small amounts of the secreted, fused protein from E. coli strains chi 1776 and HB101 bearing plasmid p147 were partially purified and assayed for biological activity by determining stimulation of [1-14C]glucose oxidation to 14CO2 in the rat epididymal fat pad assay. Guinea pig anti-insulin serum (GPAIS) was used to suppress glucose oxidation stimulated by insulin. After mild treatment with trypsin, fused protein stimulated 14CO2 production, and over 90% of this activity was suppressible by GPAIS. Untrypsinized fused protein demonstrated no GPAIS-suppressible 14CO2 production. Larger amounts of fused protein were obtained by transformation of strain PR13 of E. coli in which the yield of eukaryotic proteins is increased approx. 50-fold. A single intravenous injection of trypsinized fused protein from E. coli PR13 containing plasmid p287.47 resulted in a rapid decline in the plasma glucose levels of fasted mice, and the hypoglycemic effect persisted for at least 120 min.     

Effective, plasmid RP4-dependent replication-transposition of DNA of the transposable phage D3112 of Pseudomonas aeruginosa in a heterologous Escherichia coli system

The processes of replication and transposition of Pseudomonas aeruginosa transposable phage D3112 in cells of Escherichia coli (D3112) and E. coli (RP4::D3112) were studied. D3112 genome is a "silent cassette" ("conex-phage"--conditionally expressible) in E. coli cells incubated at 42 degrees C. Two compulsory conditions for D3112 genome expression are incubation at 30 degrees C and the presence in cells of RP4 plasmid. Processes of replication and transposition in E. coli are coupled. RP4 plasmid stimulates D3112 DNA synthesis in E. coli at least by two order of magnitude. In correspondence with this observation is the fact that when Mg2+ is present in high concentration (0.1 M) in a cultural medium, the production of mature phage is enhanced by two order of magnitude in E. coli (RP4::D3112) or in E. coli (D3112, RP4) cells, and is approx. 10(-1)-10(-2) phage per cell. No influence of Mg on phage production is observed in E. coli (D3112) cells.     

Microbial transformations in a cyclodextrin medium. Part 4. Enzyme vs microbial oxidation of cholesterol

A comparative study was conducted on two biocatalysts, resting Rhodococcus erythropolis cells and soluble cholesterol (CL) oxidase, both catalysing CL oxidation in a cyclodextrin (CD) medium. The enzyme-mediated sterol oxidation was clearly enhanced by the dimethylated -cyclodextrin (Dimeb), as in the microbial oxidation. However, the microbial transformation was subject to a larger enhancement effect (enhancement factor of approx. 6) than the enzymic one (enhancement factor of approx. 2), with respect to the corresponding transformations with no CD. Rate vs substrate concentration curves of the microbial and enzymic systems were found to be Michaelis-Menten-like with corresponding Michaelis constant (K _m) values of approx. 0.25 and 0.5 g/l. The larger Dimeb-induced effect exerted on the microbiol system was interpreted by a stronger affinity of Dimeb to the microbial cell. This CD-cell interaction was manifested through a slightly inhibited microbial growth and a limited leakage of cellular proteins and CL oxidase.Dedicated to Prof. Jochanan Blum on the occasion of his 60th birthday Correspondence to: R. Bar     

Alterations in lipopolysaccharide produced by chemostat-grown Escherichia coli O157:H7 as a function of growth rate and growth-limiting nutrient

Escherichia coli O157:H7 was grown in chemostats as continuous cultures at different controlled growth rates and under different nutrient limitations to determine the effects on lipopolysaccharide (LPS) structure. LPS from whole cells and extracted using the hot aqueous phenol method was examined by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) and by gel filtration after hydrolysis with acetic acid. At low growth rates under glucose limitation (D = 0.1 h-1, doubling time (td), approx. 416 min; or D = 0.4 h-1, td, approx. 104 min), E. coli O157 produced high molecular weight LPS identical to that previously characterized from cells grown in batch culture. At a high growth rate (D = 0.8 h-1, td, approx. 52 min), the ratio of high molecular weight LPS to low molecular weight LPS produced greatly decreased. A small amount of high molecular weight LPS, containing O-polysaccharide which lacked amino sugars, and which thus was chemically different from that previously characterized, was produced by the cells at high growth rates. The predominant form of LPS from these cells was of slightly higher molecular weight than rough LPS, probably S-R LPS, and it consistently formed aggregates on SDS-PAGE. This form of LPS was also predominant when E. coli O157 was grown under Mg2+ limitation at an intermediate growth rate (D = 0.4 h-1, td, approx. 104 min).     

Reduction of oxygen by hydrogen in cells of anaerobically grown Proteus mirabilis

Anaerobically grown cells of the enterobacteria Proteus mirabilis, Escherichia coli, Salmonella typhimurium and Enterobacter cloacae catalyse the reduction of oxygen by hydrogen (The Knallgas reaction). We have studied this reaction in detail in P. mirabilis. Oxygen at concentrations above approx. 20 μM inactivates the catalytic pathway for this reaction in a reversible way. The site of inactivation is located in the part of the pathway shared with the reduction of fumarate by hydrogen, possibly hydrogenase. Oxygen exerts its effect via the oxidation state of an unknown component in the bacteria, such that electron transfer from H₂ is blocked when this component is oxidised. We suggest that this component is identical to the regulating factor controlling hydrogen production via the formate-hydrogenlyase reaction (Krab, K., Oltmann, L.F. and Stouthamer, A.H. (1982) Biochim. Biophys. Acta 679, 51–59).     

Strain and temperature-dependent variation in the amount of BtuB polypeptide in the Escherichia coli K-12 outer membrane

Abstract The outer membrane protein BtuB of Escherichia coli K-12 is the receptor for vitamin B₁₂; it is normally present in approx. 200 copies per cell. We describe here the conditions by which BtuB was readily observed in electropherograms of outer membrane preparations. These conditions are as follows. (1) Incorporation of 8 M urea in sodium dodecyl sulfate-polyacrylamide gel systems improved detection of the polypeptide. (2) In most E. coli K-12 strains examined, BtuB was most abundant when cells were grown at 27°C. This thermoregulation of BtuB was independent of envZ and envY , two regulatory genes for outer membrane proteins.     

Microbial metabolism of amino ketones: l-1-Aminopropan-2-ol dehydrogenase and l-threonine dehydrogenase in Escherichia coli

1. A wide range of intermediary metabolites and substrate analogues have no effect on the oxidation of dl-1-aminopropan-2-ol to aminoacetone by washed-cell suspensions of Escherichia coli. Only dl-2-hydroxy-2-phenylethylamine, dl-1,3-diaminopropan-2-ol, dl-serine and l-1-(3,4-dihydroxyphenyl)-2-aminoethanol act as inhibitors. 2. Dialysed cell-free extracts of E. coli exhibit an NAD(+)-dependent dl-1-aminopropan-2-ol-dehydrogenase activity of approx. 8mmumoles of aminoacetone formed/mg. of protein/min. at the pH optimum of approx. 10. The K(m) values for the coenzyme and dl-amino alcohol are approx. 0.4 and 10.0mm respectively. A smaller peak of activity occurs at pH7.0-7.2, the K(m) for NAD(+) at pH7 being approx. 0.05mm. 3. Enzyme activity in cell-free extracts is inhibited by dl-2-hydroxy-2-phenylethylamine, dl-1-aminopropane-2,3-diol and dl-serine. dl-Phenylserine and dl-1-aminobutan-2-ol are oxidized to compounds reacting as amino ketones. 4. In fresh cell-free extracts l(+)-1-aminopropan-2-ol preparations are oxidized more rapidly than racemic or laevo-rotatory material, the d(-)-enantiomorph appearing to act as a competitive inhibitor. The K(m) for l(+)-1-aminopropan-2-ol appears to be approx. 1.5mm when highly resolved substrate preparations are used, either in the free base form or as the l(+)-tartrate salt. 5. l(+)-1-Aminopropan-2-ol dehydrogenase is a labile enzyme, and in appropriately treated extracts activity towards the d-enantiomorph is detectable and relatively higher than that towards the l-enantiomorph. 6. Optimum activity of l-threonine-dehydrogenase in cell-free extracts is exhibited at pH9.6 in the presence of NAD(+). The K(m) values for coenzyme and amino acid substrate are approx. 0.08 and 5.0mm respectively. This enzyme is distinct from 1-aminopropan-2-ol dehydrogenases on the basis of kinetic evidence, and the separation of activities by gel filtration. 7. Both l-threonine and dl-1-aminopropan-2-ol dehydrogenases are markedly inhibited by 8-hydroxyquinoline and p-chloromercuribenzoate, but only slightly by other chelating and thiol reagents. 8. E. coli is incapable of growth on simple synthetic media, containing a variety of carbon sources, when dl-1-aminopropan-2-ol is supplied as the sole source of nitrogen. It appears unlikely that the micro-organism can deaminate aminoacetone. 9. The metabolic roles of l-threonine dehydrogenase, aminoacetone and 1-aminopropan-2-ol dehydrogenases are discussed.     

Proton translocation and the respiratory nitrate reductase of Escherichia coli.

Stoicheometries and rates of proton translocation associated with respiratory reduction of NO3- have been measured for spheroplasts of Escherichia coli grown anaerobically in the presence of NO3-. Observed stoicheiometries [leads to H+/NO3- ratio; P. Mitchell (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin] were approx. 4 for L-malate oxidation and approx. 2 for succinate, D-lactate and glycerol oxidation. Measurements of the leads to H+/2e- ratio with formate as the reductant and oxygen or NO3- as the oxidant were complicated by pH changes associated with formate uptake and CO2 formation. Nevertheless, it was possible to conclude that the site of formate oxidation is on the inner aspect of the cytoplasmic membrane, that the leads to H+/O ratio for formate oxidation is approx. 4, and that the leads to H+/NO3- ratio is greater than 2. Measurements of the rate of NO3- penetration into osmotically sensitive spheroplasts demonstrated an electrogenic entry of NO3- anion. The permeability coefficient for nitrate entry at 30 degrees C was between 10(-9) and 10(-10) cm- s(-1). The calculated rate of nitrate entry at the concentration typically used for the assay of nitrate reductase (EC 1.7.99.4) activity was about 0.1% of that required to support the observed rate of nitrate reduction by reduced Benzyl Viologen. Measurements of the distribution of nitrate between the intracellular and extracellular spaces of a haem-less mutant, de-repressed for nitrate reductase but unable to reduce nitrate by the respiratory chain, showed that, irrespective of the presence or the absence of added glucose, nitrate was not concentrated intracellularly. Osmotic-swelling experiments showed that the rate of diffusion of azid anion across the cytoplasmic membrane is relatively low in comparison with the fast diffusion of hydrazoic acid. The inhibitory effect of azide on nitrate reductase was not altered by treatments that modify pH gradients across the cytoplasmic membrane. It is concluded that the nitrate-reducing azide-sensitive site of nitrate reductase is located on the outer aspect of the cytoplasmic membrane. The consequences of this location for mechanisms of proton translocation driven by nitrate reduction are discussed, and lead to the proposal that the nitrate reductase of the cytoplasmic membrane is vectorial, reducing nitrate on the outer aspect of the membrane with 2H+ and 2e- that have crossed from the inner aspect of the membrane.     

Quantitative effects of unsaturated fatty acids in microbial mutants. VII. Influence of the acetylenic bond location on the effectiveness of acyl chains.

The ability of a series of 18 carbon acetylenic fatty acids to fulfill the unsaturated fatty acid requirements of Escherichia coli and Saccharomyces cerevisiae was investigated. Despite their high melting points (greater than 40 degrees C), several isomers of the acetylenic fatty acids were as efficient or more efficient in supporting growth than the analogous fatty acid having a cis-double bond. The efficiencies of the different positional isomers in supporting cell proliferation varied from essentially 0 cells per fmol for the 2-5 and 13-17 isomers to high values when the acetylenic bond was near the center of the chain: e.g. 45 E. coli and 5.5 S. cerevisiae cells/fmol for the 10 isomer. A striking ineffectiveness of the 9 isomer was observed with E. coli. The 7, 8 and 10 isomers were at least 10-fold more efficient than any of the other positional isomers in supporting the growth of E. coli. In contrast, the 9 isomer was among the most effective acetylenic fatty acids tested with the yeast mutant. Chromatographic analysis of the extracted lipids indicated that each of the acetylenic isomers tested (except delta2 and delta3) could be esterified by the prokaryotic and eukaryotic microorganisms. The content of unsaturated plus cyclopropane acids observed when growth ceased in E. coli cultures supplemented with growth-limiting concentrations of the acetylenic fatty acids ranged from approx. 15 mol% for the 8 isomer to approx. 35 mol% for the 14 and 17 isomers. The 8-11 isomers were observed to be esterified predominantly at the two position in phosphatidylethanolamine of E. coli and in phosphatidylcholine of S. cerevisiae.     

Relation between microsecond reduction kinetics of photooxidized chlorophyll aII (P-680) and photosynthetic water oxidation

The reduction phases of chlorophyll a⁺_II (P-680⁺) in the microsecond range have been studied in O₂-evolving Photosystem II particles from Synechococcus sp. and in spinach subchloroplasts. (1) In selected Photosystem II preparations only approx. 15% of chlorophyll a⁺_II is reduced under repetitive excitation in the microsecond time-range (approx. 85% are reduced in the nanosecond time-range). (2) The size of the microsecond fraction varies as a function of the flash number given to dark-adapted samples, suggesting a correlation to the oxidation states of the O₂-evolving complex (S-states). The oscillatory pattern closely follows the concentration of S₂ + S₃. (3) The microsecond decay can be deconvoluted into three exponential phases with half-life times of approx. 5, 35 and 200 μs. It is the amplitude of the 35 μs phase which depends on S₂ + S₃. Therefore, the 35 μs phase (approx. 10% under repetitive excitation) is connected with water oxidation. (4) Considerably higher values of the μs fraction (up to 50%) reported in former publications were probably due to Photosystem II centers which were inactive in O₂ evolution.     

Microbial metabolism of amino ketones. Aminoacetone formation from 1-aminopropan-2-ol by a dehydrogenase in Escherichia coli

1. Washed-cell suspensions of Escherichia coli, incubated at the optimum pH of 6.4 and with a saturating substrate concentration of approx. 10mm, convert dl-1-aminopropan-2-ol into aminoacetone at a rate of approx. 4.0mmumoles/mg. dry wt. of cells/min. at 30 degrees . 2. Mg(2+), Mn(2+), Co(2+), Zn(2+), Ca(2+), K(+) and NH(4) (+), as sulphates, and EDTA have no effect on this rate, although Cu(2+) inhibits and Fe(2+) activates to some extent. 3. Conditions of growth markedly affect the rate of aminoacetone production by cell suspensions. 4. Dialysed cell-free extracts of E. coli exhibit 1-aminopropan-2-ol-dehydrogenase activity, the enzyme having optimum activity at pH7.0, a requirement for NAD(+) and K(+), and a K(m) for the amino alcohol substrate of 0.8mm, calculated for a single enantiomorph. 5. Under optimum conditions 1-aminopropan-2-ol dehydrogenase forms aminoacetone at rate of approx. 3.0mmumoles/mg. of protein/min. at 37 degrees . The enzyme is only slightly inhibited by dl-3-hydroxybutyrate and dl-2-hydroxy-2-phenylethyl-amine. 6. l-Threonine-dehydrogenase activity is exhibited by both whole cells and cell-free extracts. Whole cells produce aminoacetone from l-threonine more slowly than they do from dl-1-aminopropan-2-ol, whereas the situation is reversed in cell-free extracts. Both kinetic evidence, and the fact that synthesis of 1-aminopropan-2-ol dehydrogenase, but not of threonine dehydrogenase, is repressed by compounds such as glucose and pyruvate, provide evidence that the amino alcohol is oxidized by a specific enyme. 7. The metabolic role of 1-aminopropan-2-ol dehydrogenase is discussed.     

Mutagenicity of secondary oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate)

8-Oxo-7,8-dihydroguanine (8-hydroxyguanine) is oxidized more easily than normal nucleobases, which can produce spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). These secondary oxidation products of 8-oxo-7,8-dihydroguanine are highly mutagenic when formed within DNA. To evaluate the mutagenicity of the corresponding oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate) in the nucleotide pool, Escherichia coli cells deficient in the mutT gene were treated with H(2)O(2), and the induced mutations were analyzed. Moreover, the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh were also introduced into competent E. coli cells. The H(2)O(2) treatment of mutT E. coli cells resulted in increase of G:C → T:A and A:T → T:A mutations. However, the incorporation of exogenous Sp and Gh 2'-deoxyribonucleotides did not significantly increase the mutation frequency. These results suggested that the oxidation product(s) of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate induces G:C → T:A and A:T → T:A mutations, and that the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh exhibit quite weak mutagenicity, in contrast to the bases in DNA.     

The enzymic reduction and kinetics of oxidation of cytochrome b-245 of neutrophils.

1. The absorption coefficient of human neutrophil plasma-membrane reduced-minus-oxidized cytochrome b-245 was determined [delta epsilon (mM; 559-540 nm) = 21.6 cm-1]. 2. Neutrophil polymorphonuclear leucocytes (neutrophils) were prepared from human, ox, horse and pig blood. In each case plasma-membrane fractions were found to contain low-potential cytochrome b. When membranes from horse neutrophils were incubated anaerobically with either NADH or NADPH the cytochrome b became reduced. Prior stimulation of the cells with phorbol myristate acetate did not increase the rate or extent of cytochrome b reduction in isolated membranes, but did increase both the rate and extent of reduction by NADPH in Triton-treated cells. 3. A cytochrome b was present also in the specific granule fraction of human neutrophils. Its Em (pH 7.0) was found to be -248 mV, very similar to that of the plasma-membrane cytochrome b. 4. The rate of oxidation of reduce cytochrome b-245 by air-saturated buffer, was determined by using stopped-flow techniques. In intact membranes t 1/2 for oxidation was 4.7 ms. This rate is sufficiently rapid to support the view that cytochrome b-245 is the oxidase in the respiratory burst of neutrophils. 5. Plasma-membrane cytochrome b of human neutrophils formed a complex with CO. At room temperature and 1 atm of CO approx. 40% of the cytochrome formed a complex; approx. 60% binding was measured at the increased concentration of dissolved CO achieved at 5 degrees C. The concentration of CO giving 50% binding was 1.18 mM.     

Numbers and transported state of Escherichia coli in runoff direct from fresh cowpats under simulated rainfall

AIMS: To investigate the number of Escherichia coli in runoff derived directly from fresh cowpats and to determine if the E. coli are attached to dense particles, in flocs or as individual cells. METHODS AND RESULTS: Three cowpats were collected monthly from the same farm for 13 months and the number of E. coli in them estimated. A rainfall simulator was used to generate runoff from the individual cowpats, which was fractioned to determine the transported state of any E. coli present. The number of E. coli in the cowpat runoff was highly variable and was strongly correlated with the number of E. coli in the cowpat. Only a small percentage (approx. 8%) of the E. coli in runoff were attached to dense (>1.3 g ml(-1)) particles and there was no evidence of flocculation of the cells. CONCLUSIONS: Escherichia coli in runoff from cowpats are transported predominantly as individual cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Mitigation strategies to reduce the number of faecal bacteria in overland flow from agricultural land need to be designed to trap single bacterial cells.     

The properties of adenosine triphosphatase from exponential and synchronous cultures of Alcaligenes eutrophus H16

The properties of Alcaligenes eutrophus ATPase (adenosine triphosphatase) were investigated by using subcellular fractions prepared from cells growing in exponential and synchronous cultures. Both the soluble and membrane-bound forms of the ATPase were inhibited non-competitively (K(i) 142mum) by Nbf-Cl (4-chloro-7-nitrobenzofurazan), whereas only the membrane-bound enzyme was inhibited (non-competitive; K(i) 750mum) by NN'-dicyclohexylcarbodi-imide. Neither the activity of the ATPase nor its sensitivity to these two inhibitors varied during exponential growth. However, marked variations in ATPase activity were observed during synchronous growth, which were characterized by maxima at approx. 0.4 and 0.9 of a cell cycle and minima at approx. 0.1 and 0.6 of a cycle. Sensitivity to Nbf-Cl and NN'-dicyclohexylcarbodi-imide also varied during the cell cycle; maximum inhibition by the former occurred at approx. 0.4 and 0.9 of a cell cycle, whereas maximum inhibition by the latter was located at approx. 0.1 and 0.6 of a cell cycle. Proton conductance by whole cells was also periodic during the cell cycle, the lowest rates occurring at approx. 0.15 and 0.55 of a cycle and the highest rates at approx. 0.4 and 0.9 of a cycle, but -->H(+)/O quotients for the oxidation of endogenous substrates remained relatively constant and indicated the presence of four proton-translocating respiratory segments throughout the cell cycle. These results are discussed in terms of ATPase and respiratory-chain structure and function during the cell cycle of Alcaligenes eutrophus.     

Potentiometric titration of cytochrome-bo type quinol oxidase of Escherichia coli: evidence for heme-heme and copper-heme interaction

The cytochrome-bo quinol oxidase of Escherichia coli contains a high-spin b-type heme (cytochrome o), a low-spin b-type heme (cytochrome b) and copper. The EPR signal from cytochrome o is axial high spin and when titrated potentiometrically gives a bell-shaped curve. The low-potential side of this curve (Em7 approx. 160 mV) corresponds to the reduction/oxidation of the cytochrome. The high-potential side (Em7 approx. 350 mV) is proposed to be due to reduction/oxidation of a copper center; in the CuII form tight cytochrome o-copper spin coupling results in a net even spin system and loss of the EPR spectrum. Optical spectra of the alpha-bands of the reduced cytochromes at 77 K show that cytochrome b has its maxima at 564 nm when cytochrome o is oxidized but that this shifts to 561 nm when cytochrome o (max. 555 nm) is reduced. Both a heme-copper (cytochrome o-CuII) and a heme-heme (cytochrome o-cytochrome b) interaction are indicated in this quinol oxidase. These results indicate that cytochrome-bo quinol oxidase has a binuclear heme-copper catalytic site and suggest striking structural similarity to subunit I of the cytochrome aa3 system.     

Does polymyxin B nonapeptide increase outer membrane permeability in antibiotic supersensitive enterobacterial mutants?

Abstract The outer-membrane-disorganizing peptide (polymyxin B nonapeptide; PMBN) was able to sensitize even "antibiotic supersensitive" enterobacterial mutants to hydrophobic antibiotics. This resulted in an extreme sensitivity. The mutants included the "deep rough" lipopolysaccharide mutants, as well as the acrA mutant of Escherichia coli and the "class A, B, and C mutants of Salmonella typhimurium . Sensitization factors of approx. 30 or more were found for most antibiotics. Even minimum inhibitory concentrations as low as approx. 0.5 ng/ml (rifampicin), 1.5 ng/ml (erythromycin), 2 ng/ml (fusidic acid), 6 ng/ml (novobiocin), and 30 ng/ml (clindamycin) were achieved in the presence of 30 μg/ml of PMBN. The finding indicates that the mechanisms which mediate the increase in hydrophobic diffusion are different but synergistic in the mutants and in the PMBN-grown cells.     

Isolation of a clone which induces expression of the gene encoding the human tumor necrosis factor receptor.

Tumor necrosis factor (TNF) is a cytokine with pleiotropic effects upon cell growth, inflammation and immunologic responsiveness. High-affinity TNF receptors (TNFRs) of 55 and 75 kDa are found in many cell types. Using an Epstein-Barr virus (EBV)-based mammalian expression library, we have isolated a clone from human lymphoblastoid transfectants that induces overexpression of the TNFR-encoding gene (TNFR). Transfectants overproducing the TNFR were isolated by multiple rounds of sorting on a fluorescence-activated cell sorter using fluorescent TNF ligand binding as the selection procedure. Among the sorted transfectants were cells producing approx. 150,000 receptors per cell (Kd of approx. 1 nM). These cells have multiple copies of the TNFR gene present as extrachromosomal plasmids. These cells also overproduced the mRNA for TNFR. Low-Mr EBV episomes were isolated from these overproducing cells and used to transform Escherichia coli. One of the colonies isolated contained a plasmid encoding a portion of the noncoding region of the TNFR gene. Transfection of human lymphoblastoid cells with this DNA gave rise to high-level production of TNFR. Fluorescent TNF bound to these transfectants is fully and specifically displaced by an excess of TNF. The rescued clone contains approx. 10 kb of human genomic DNA including the 3'-untranslated region of TNFR and several Alu sequences; apparently during the selection procedure in human cells, recombination occurred to rescue a portion of the TNFR gene. Transient transfection was used to narrow down the region responsible for TNFR induction to 5.2 kb. The mechanism by which this clone induces TNFR expression has not been determined.     

大肠杆菌表达的人重组IL-3的纯化

本文继大肠杆菌表达含凝血酶切点的人重组IL-3融合蛋白成功的基础上进一步探讨了天然型rhIL-3的纯化。超声破碎细菌细胞得包涵体,经洗涤处理可使包涵体纯度达90％,用8mol/L尿素变性溶解包涵体沉淀后直接稀释复性,再超滤浓缩、凝血酶消化,释放天然型rhIL-3。经DEAE Sepharose Fast Flow和Sepharcyl-100 HR层析得到天然型IL-3,纯度达96％,回收率20％以上,具有刺激正常人骨髓细胞形成集落的活性。本实验为大批量重组IL-3的生产创造了条件。     

High-level expression of a functional human fibrinogen gamma chain in Escherichia coli

Human fibrinogen gamma chain has been expressed intact at high levels in Escherichia coli. The construction of the expression plasmid, p253, is described. Synthesis of the recombinant protein is isopropyl-beta-D-thiogalactopyranoside-dependent and is driven by the tac promoter. Western analysis of E. coli lysates demonstrates a novel protein of approx. 45 kDa which cross-reacts with antisera to human fibrinogen gamma chains. The protein is not soluble in common E. coli lysis buffers and becomes soluble in 6 M guanidine.HCl or 6 M urea. Initial insolubility is due to interchain disulfide bond formation and to noncovalent interactions. Induced cells examined by phase-contrast microscopy contain dense inclusion bodies. A known function of the gamma chains of human fibrinogen is the clumping of Staphylococcus aureus Newman D2C cells [Hawiger et al., Biochemistry (1982) 1407-1413]. We demonstrate that suspensions of recombinant gamma chains retain the ability to clump cells from this strain of S. aureus.