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Biomedical subjects

G Georgiou

Publications and source records attributed to G Georgiou.

At least 73 records · Page 4Linked to original sources
PubMed1996

Degradation of Trichloroethylene by Methanol-Grown Cultures of Methylosinus trichosporium OB3b PP358.

A soluble methane monooxygenase-constitutive mutant strain of Methylosinus trichosporium OB3b, strain PP358, was grown with methanol as the carbon source, and the kinetics of trichloroethylene (TCE) degradation were determined. PP358 exhibited high TCE degradation rates under both oxygen- and carbon-limiting conditions. The optimal pseudo first-order rate constant for TCE was comparable to the values measured for cells grown with methane. We found that growth under oxygen-limiting conditions results in increased accumulation of polyhydroxybutyrate, which in turn correlates with higher transformation capacities for TCE. It was also shown that methanol inhibits TCE degradation only at high concentrations. Thus, methanol-grown cultures of PP358 represent an efficient system for the biodegradation of chlorinated hydrocarbons.

Journal Article↗
PubMed1995

Experience with warm blood cardioplegia in 480 patients.

From 1 March 1992 too 31 July 1993 (17 months), 480 consecutive patients underwent various open-heart procedures under anterograde (83 patients) or retrograde (397 patients) or retrograde (397 patients) continuous warm blood cardioplegia. Some 352 patients (73.3%) had isolated coronary artery bypass grafts (CABG) and 117 (24.3%) had valve replacement either isolated (96) or in combination with other operations (21). Two patients had CABG and ventricular aneurysmectomy, eight had correction of congenital defects, and one had resection of left atrial myxoma. The 30-day postoperative mortality rate was 2.9% (14 deaths). In four patients the cause of death was not cardiogenic. An intra-aortic balloon was used in 11 patients following CABG (3.1%) with six survivors. Perforation of the coronary sinus occurred in one patient. Perioperative myocardial infarction was observed in 5.6% of patients after CABG. No myocardial infarction occurred after valve replacement. Phrenic nerve injury and wound infection were not observed. These results indicate that warm blood cardioplegia, especially when delivered retrogradely, provides excellent myocardial protection of both ventricles during various open-heart procedures.

Adult↗
PubMed1994

The expression of recombinant proteins on the external surface of Escherichia coli. Biotechnological applications.

The expression of recombinant proteins on the external surface of Gram-negative bacteria is expected to open the way for a number of significant biotechnological applications, including the development of live bacterial vaccines, the production of whole cell adsorbents, the preparation of whole cell catalysts, and the display and selection of peptide and antibody libraries. We have developed a fusion protein system for the production of active recombinant proteins on the surface of Escherichia coli. Using this system we have expressed beta-lactamase, the Cellulomonas fimi exoglucanase Cex as well as its cellulose binding domain, and an antidigoxin single chain Fv antibody fragment on the cell surface. Recently we have begun to explore some of the potential applications for cell-surface expression.

Bacterial Outer Membrane Proteins↗
PubMed1994

The folding of bovine pancreatic trypsin inhibitor in the Escherichia coli periplasm.

PreOmpA-bovine pancreatic trypsin inhibitor (BPTI) (Goldenberg, D. P. (1988) Biochemistry 27, 2481-2489) was expressed in Escherichia coli, and the folding pathway of the mature protein in the periplasmic space was analyzed by pulse-chase experiments. Folding intermediates were trapped with iodoacetamide, immunoprecipitated with antisera specific for either the reduced or the native protein, and resolved by electrophoresis. In vivo, native BPTI formed with a half-life of 7 min which is 3-fold faster than the optimal in vitro folding rate in growth media supplemented with low molecular weight disulfides. The measured in vivo half-life includes the time required for translocation and processing by leader peptidase and therefore represents the lower limit for the actual folding rate in the cell. In addition to the native species, two-disulfide intermediates accumulated in the cell at appreciable levels and did not chase to the native species for at least 20 min. We found that the folding of BPTI in E. coli was absolutely dependent on DsbA, a protein which accelerates the formation of disulfide bonds in the periplasm. In a dsbA mutant strain, trace amounts of oxidized BPTI could be detected only in cultures grown under strongly oxidizing conditions. In wild-type cells, the addition of different concentrations of GSH/GSSG or oxidized dithiothreitol did not affect the kinetics of BPTI oxidation by DsbA. Finally, even though the folding of BPTI in vitro decreases by almost 10-fold/unit pH decrease, folding in cells grown at pH 6.0 was only marginally slower than folding in cells grown at neutral pH, despite the fact that the pH of the periplasmic space varies in response to the extracellular fluid.

Animals↗
PubMed1994

Folding and aggregation of TEM beta-lactamase: analogies with the formation of inclusion bodies in Escherichia coli.

The enzyme TEM beta-lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM beta-lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0-1.4 M) concentrations of guanidium chloride (GdmCl). This species exhibits a large increase in bis-1-anilino-8-naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces. When TEM beta-lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0-1.5 M range. It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates. We propose that the aggregation of TEM beta-lactamase involves the association of a compact state having partially exposed hydrophobic surfaces. This hypothesis is consistent with our recent findings that TEM beta-lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7:131-136). Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose. These conditions exert a similar effect on the formation of inclusion bodies in vivo.

Anilino Naphthalenesulfonates↗
PubMed1994

Construction and characterization of a set of E. coli strains deficient in all known loci affecting the proteolytic stability of secreted recombinant proteins.

Even though secretion offers numerous advantages for the production of proteins in Escherichia coli, the expression of many heterologous proteins is severely limited by degradation in the periplasmic space. We found that mutations in rpoH, the RNA polymerase sigma factor responsible for heat shock protein synthesis, affect the stability of heterologous secreted proteins. A particularly dramatic increase in expression was further observed in rpoH degP double mutants. To minimize proteolytic degradation, we constructed a family of 25 isogenic strains deficient in all known cell envelope proteases (DegP, Protease III, Tsp(Prc), and OmpT), as well as the rpoH15 mutant allele, and characterized their growth in both shake flasks and fermentors. The availability of this set of strains permits the selection of a suitable host based on the optimal combination between the optimum reduction in protease activity and acceptable growth properties.

Chromosome Mapping↗
PubMed1994

Secondary structure characterization of beta-lactamase inclusion bodies.

The secondary structure of proteins in E. coli inclusion bodies was investigated via Raman spectroscopy. Inclusion bodies were purified from cells expressing different forms of RTEM beta-lactamase and grown at either 37 or 42 degrees C. All of the solid phase inclusion body samples examined gave amide I band spectra that were perturbed from that of the native, purified protein in both solution and powder forms; secondary structure estimates indicated significant decreases in alpha-helix and increases in beta-sheet contents in the inclusion body samples. The structure estimates for inclusion bodies isolated from 37 degrees C cultures were similar, regardless of aggregate localization in the E. coli cytoplasmic or periplasmic spaces or beta-lactamase precursor content. Inclusion bodies obtained from 42 degrees C cells exhibited a further reduction of alpha-helix and augmentation of beta-sheet contents relative to those from 37 degrees C cultures. These results are consistent with the paradigm for inclusion body formation via the self-association of intra-cellular folding intermediates having extensive secondary structure content. Further, the overall secondary structure content of inclusion bodies is not significantly affected by subcellular compartmentalization, but may be altered at increased temperatures.

Escherichia coli↗
PubMed1994

Structural and immunological characterization of a biosurfactant produced by Bacillus licheniformis JF-2.

Bacillus licheniformis JF-2 produces a very active biosurfactant under both aerobic and anaerobic conditions. We purified the surface-active compound to homogeneity by reverse-phase C18 high-performance liquid chromatography and showed that it is a lipopeptide with a molecular weight of 1,035. Amino acid analysis, fast atom mass and infrared spectroscopy, and, finally, 1H, 13C, and two-dimensional nuclear magnetic resonance demonstrated that the biosurfactant consists of a heterogeneous C15 fatty acid tail linked to a peptide moiety very similar to that of surfactin, a lipopeptide produced by Bacillus subtilis. Polyclonal antibodies were raised against surfactin and shown to exhibit identical reactivity towards purified JF-2 lipopeptide in competition enzyme-linked immunosorbent assays, thus providing further evidence for the structural similarity of these two compounds. Under optimal conditions, the B. licheniformis JF-2 biosurfactant exhibits a critical micelle concentration of 10 mg/liter and reduces the interfacial tension against decane to 6 x 10(-3) dyne/cm, which is one of the lowest interfacial tensions ever reported for a microbial surfactant.

Amino Acid Sequence↗
PubMed1993

Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface.

We have expressed a single chain Fv (scFv) antibody fragment, consisting of the variable heavy and variable light domains from two separate anti-digoxin monoclonal antibodies, on the external surface of Escherichia coli by fusing it to an Lpp-OmpA hybrid previously shown to direct heterologous proteins to the cell surface. This scFv fusion was expressed at a high level and was shown to bind the hapten with high affinity and specificity. Whole cell ELISAs, fluorescence microscopy, protease sensitivity, and flow cytometry all confirmed that the scFv was anchored on the outer membrane and was accessible on the surface. Utilizing fluorescence-activated cell sorting, we were able to specifically enrich scFv-producing cells from a 10(5)-fold excess of control cells in only two steps. The expression of antibody fragments on the surface of E. coli is being evaluated as an attractive method for the in vitro production and selection of useful antibody fragments.

Amino Acid Sequence↗
PubMed1993

Practical applications of engineering gram-negative bacterial cell surfaces.

The recent development of systems for the expression of heterologous proteins on the surface of Gram-negative bacteria has stimulated considerable interest in practical applications. Areas in which surface expression is particularly important include the development of live bacterial vaccines, the display and selection of peptide and antibody libraries, the production of whole cell adsorbents, and the preparation of microbial biocatalysts.

Animals↗
PubMed1993

Specific adhesion and hydrolysis of cellulose by intact Escherichia coli expressing surface anchored cellulase or cellulose binding domains.

The entire Cex exoglucanase from Cellulomonas fimi and the Cex Cellulose Binding Domain (CBDCex) were expressed in Escherichia coli as fusions to an Lpp-OmpA hybrid which had been shown earlier to direct a heterologous protein to the cell surface. Both Cex and CBDCex were readily localized on the cell surface and could be detected by immunofluorescence microscopy, whole cell ELISAs and functional assays. In cells expressing the entire Cex, about 90% of the total cellobiose hydrolase activity was anchored on the external side of the outer membrane and was susceptible to protease (papain) added in the extracellular fluid. Cells expressing either Cex or CBDCex bound tightly and rapidly to cellulosic materials such as cotton fibers. This property can be exploited for the preparation of immobilized microbial biocatalysts via adsorption to cellulose and for cell separation through specific agglutination on inexpensive cellulosic materials. In addition, our results demonstrate the general utility of fusions to lpp-ompA for the efficient display of proteins and the engineering of the surface topology of Gram-negative bacteria.

Actinomycetales↗
PubMed1993

Phenotypic characterization of copper-resistant mutants of Methylosinus trichosporium OB3b.

Cultures of Methylosinus trichosporium OB3b grown in the presence of very low concentrations of copper synthesize a soluble methane monooxygenase (sMMO) that efficiently catalyzes the oxidation of trichloroethylene and other organic pollutants. Recently, we isolated five M. trichosporium OB3b mutants that express sMMO activity when grown in the presence of elevated copper concentrations (P.A. Phelps, S. K. Agarwal, G. E. Speitel, Jr., and G. Georgiou, Appl. Environ. Microbiol. 58:3701-3708, 1992). Here we show that, in contrast to the results for the wild-type cells, the addition of copper to mutant cultures grown on methane and nitrate as the nitrogen source has no noticeable effect on the growth rate and sMMO expression. In vitro experiments indicated that the copper-resistant phenotype does not arise from an increased stability of sMMO to copper deactivation. Furthermore, the mutant cultures exhibit altered speciation of copper in the extracellular fluid and have substantially decreased levels of cell-associated copper. On the basis of these results, we propose that the mutant phenotype arises from defects in copper uptake and metabolism rather than from changes in sMMO expression or enzyme stability.

Biodegradation, Environmental↗
PubMed1992

Transport and anchoring of beta-lactamase to the external surface of Escherichia coli.

The outer membrane of Gram-negative bacteria presents an effective barrier that restricts the release of proteins from the cell. Virtually all extracellular proteins of Gram-negative bacteria are exported by specialized systems requiring the action of several gene products. We have constructed a tripartite fusion consisting of (i) the signal sequence and first nine N-terminal amino acids of the mature major Escherichia coli lipoprotein, (ii) amino acids 46-159 of the outer membrane protein OmpA, and (iii) the complete mature beta-lactamase (EC 3.5.2.6) sequence. This protein had an enzymatically active beta-lactamase and was found predominantly in the outer membrane. Immunofluorescence microscopy, the accessibility of the fusion protein to externally added proteases, and the rates of hydrolysis of nitrocefin and penicillin G by whole cells demonstrated that a substantial fraction (20-30%) of the beta-lactamase domain of the fusion protein was exposed on the external surface of E. coli. In cells grown at 24 degrees C the localization of beta-lactamase on the cell surface was almost quantitative (greater than 80% of the enzymatically active protein was exposed to the extracellular fluid) as determined by nitrocefin and penicillin G hydrolysis and trypsin accessibility. These results demonstrated that a soluble protein, beta-lactamase, can be transported through--and become anchored on--the outer membrane by fusion to the proper targeting and localization signals.

Amino Acid Sequence↗
PubMed1992

Surface-active compounds from microorganisms.

Microbial surfactants are a structurally diverse group of compounds consisting of hydrophilic and hydrophobic domains and which partition preferentially at interfaces. Biosurfactants are of increasing interest commercially as substitutes for synthetic surfactants particularly for environmental applications. This article discusses recent progress in the genetic and biochemical analysis of biosurfactant synthesis as well as the current status of fermentation technologies.

Bacteria↗
PubMed1992

Methylosinus trichosporium OB3b Mutants Having Constitutive Expression of Soluble Methane Monooxygenase in the Presence of High Levels of Copper.

The methanotrophic bacterium Methylosinus trichosporium OB3b is unusually active in degrading recalcitrant haloalkanes such as trichloroethylene (TCE). The first and rate-limiting step in the degradation of TCE is catalyzed by a soluble methane monooxygenase (sMMO). This enzyme is not expressed when the cells are grown in the presence of copper at concentrations typically found in polluted groundwater. Under these conditions, M. trichosporium OB3b expresses a particulate form of the enzyme (pMMO), which has a narrow substrate specificity and does not degrade TCE at any significant rate. We have isolated M. trichosporium OB3b mutants that are deficient in pMMO and express sMMO constitutively in the presence of elevated concentrations of copper. One mutant (PP358) exhibited a TCE degradation rate which was almost twice as high as that of the wild-type strain grown under optimal conditions (without copper). All of the mutants lost the ability to express pMMO activity and to form stacked intracellular membranes characteristic of wild-type cells expressing pMMO.

Journal Article↗