Biomedical subjects

G Georgiou

Publications and source records attributed to G Georgiou.

At least 55 records · Page 3Linked to original sources

PubMed1999

In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site.

For the first time, each specificity determining residue (SDR) in the binding site of an antibody has been replaced with every other possible single amino acid substitution, and the resulting mutants analyzed for binding affinity and specificity. The studies were conducted on a variant of the 26-10 antidigoxin single chain Fv (scFv) using in vitro scanning saturation mutagenesis, a new process that allows the high throughput production and characterization of antibody mutants [Burks,E.A., Chen,G., Georgiou,G. and Iverson,B.L. (1997) Proc. Natl Acad. Sci. USA, 94, 412-417]. Single amino acid mutants of 26-10 scFv were identified that modulated specificity in dramatic fashion. The overall plasticity of the antibody binding site with respect to amino acid replacement was also evaluated, revealing that 86% of all mutants retained measurable binding activity. Finally, by analyzing the physical properties of amino acid substitutions with respect to their effect on hapten binding, conclusions were drawn regarding the functional role played by the wild-type residue at each SDR position. The reported results highlight the value of in vitro scanning saturation mutagenesis for engineering antibody binding specificity, for evaluating the plasticity of proteins, and for comprehensive structure-function studies and analysis.

Amino Acids↗

PubMed1999

Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface.

Polypeptide library screening technologies are critically dependent upon the characteristics of the expression system employed. A comparative analysis of the lpp-lac, tet and araBAD promoters was performed to determine the importance of tight regulation and expression level in library screening applications. The surface display of single-chain antibody (scFv) in Escherichia coli as an Lpp-OmpA' fusion was monitored using a fluorescently tagged antigen in conjunction with flow cytometry. In contrast to the lpp-lac promoter, both tet and araBAD promoters could be tightly repressed. Tight regulation was found to be essential for preventing rapid depletion of library clones expressing functional scFv and thus for maintaining the initial library diversity. Induction with subsaturating inducer concentrations yielded mixed populations of uninduced and fully induced cells for both the tet and araBAD expression systems. In contrast, homogeneous expression levels were obtained throughout the population using saturating inducer concentrations and could be adjusted by varying the induction time and plasmid copy number. Under optimal induction conditions for the araBAD system, protein expression did not compromise either cell viability or library diversity. This expression system was used to screen a library of random scFv mutants specific for digoxigenin for clones exhibiting improved hapten dissociation kinetics. Thus, an expression system has been developed which allows library diversity to be preserved and is generally applicable to the screening of E. coli surface displayed libraries.

Antibodies↗

PubMed1998

Molecular determinants of bacterial adhesion monitored by atomic force microscopy.

Bacterial adhesion and the subsequent formation of biofilm are major concerns in biotechnology and medicine. The initial step in bacterial adhesion is the interaction of cells with a surface, a process governed by long-range forces, primarily van der Waals and electrostatic interactions. The precise manner in which the force of interaction is affected by cell surface components and by the physiochemical properties of materials is not well understood. Here, we show that atomic force microscopy can be used to analyze the initial events in bacterial adhesion with unprecedented resolution. Interactions between the cantilever tip and confluent monolayers of isogenic strains of Escherichia coli mutants exhibiting subtle differences in cell surface composition were measured. It was shown that the adhesion force is affected by the length of core lipopolysaccharide molecules on the E. coli cell surface and by the production of the capsular polysaccharide, colanic acid. Furthermore, by modifying the atomic force microscope tip we developed a method for determining whether bacteria are attracted or repelled by virtually any biomaterial of interest. This information will be critical for the design of materials that are resistant to bacterial adhesion.

Bacterial Adhesion↗

PubMed1998

Cytogenetic changes in benign proliferative and nonproliferative lesions of the breast.

Cytogenetic analysis of short-term cultures from 69 cases of fibrocystic breast changes and 10 samples of normal mammary tissue revealed clonal chromosome aberrations in six fibrocystic lesions. All the histologically normal tissue samples had a normal karyotype. The frequency of cytogenetically abnormal cases seems to correlate with the degree of histopathologic changes of the tissue; nonproliferative lesions may have clonal chromosome alterations, but at a low frequency. Whether women with karyotypically altered fibrocystic "disease" have a higher risk of developing invasive breast cancer, compared with women without microscopically visible genetic anomalies in fibrocystic lesions, remains unknown.

Adult↗

PubMed1998

Antibody affinity maturation using bacterial surface display.

A quantitative system for screening combinatorial single-chain Fv (scFv) antibody libraries was developed utilizing surface display on Escherichia coli and fluorescence-activated cell sorting (FACS). This system was employed to isolate clones with high-affinity to a fluorescently-labeled hapten from libraries constructed by randomizing heavy and light-chain residues in the anti-digoxin 26-10 derived antibody, scFv(dig). The use of flow cytometry enabled the detection of rare library members directly in heterogeneous populations and the optimization of selection conditions prior to sorting. A heavy-chain mutant having wild-type affinity (KD = 0.91+/-0.22 nM) and an expected representation frequency of less than 1 x 10(6), was selected to homogeneity after three rounds utilizing increasingly stringent selection conditions. The isolated clone possessed two distinct point mutations relative to the wild-type DNA sequence, yet still coded for the wild-type amino acid sequence, suggesting that the wild-type residues may be optimal at the randomized positions. An affinity improved clone (KD = 0.30+/-0.05 nM), having a dissociation constant approximately threefold lower than the wild-type antibody, was isolated from a smaller light-chain library in a single sorting step. Flow cytometry was shown to be a simple and rapid method for the determination of the relative hapten dissociation rate constants of selected clones without requiring subcloning. The relative rate constants estimated by FACS were confirmed by producing the scFv antibodies in soluble form and measuring hapten binding kinetics by surface plasmon resonance (SPR). These results demonstrate that E.coli surface display, coupled with quantitative selection and analysis using FACS, has the potential to become a powerful tool for rapid isolation and characterization of desirable mutants from large polypeptide libraries.

Antibody Affinity↗

PubMed1998

Expression of active human tissue-type plasminogen activator in Escherichia coli.

The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 microgram of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins in E. coli without the need for in vitro refolding.

Cloning, Molecular↗

PubMed1998

Identification of OmpT as the protease that hydrolyzes the antimicrobial peptide protamine before it enters growing cells of Escherichia coli.

The influence of extracytoplasmic proteases on the resistance of Escherichia coli to the antimicrobial peptide protamine was investigated by testing strains with deletions in the protease genes degP, ptr, and ompT. Only DeltaompT strains were hypersusceptible to protamine. This effect was abolished by plasmids carrying ompT. Both at low and at high Mg2+ concentrations, ompT+ strains cleared protamine from the medium within a few minutes. By contrast, at high Mg2+ concentrations, protamine remained present for at least 1 h in the medium of an ompT strain. These data indicate that OmpT is the protease that degrades protamine and that it exerts this function at the external face of the outer membrane.

Escherichia coli↗

PubMed1998

Evaluating the interaction of bacteria with biomaterials using atomic force microscopy.

Bacterial infection of biomaterials represents one of the most important reasons for the failure of transdermal or implanted medical devices. The first and least understood step in biomaterial-associated infections is the initial interaction between bacteria and a surface. This initial interaction can be either attractive or repulsive depending on the physiochemical nature of the biological and synthetic surfaces, as well as the properties of the interstitial fluid. We have shown that atomic force microscopy (AFM) can be employed as an exquisitely sensitive and versatile tool for quantifying the interaction between bacteria and surfaces in physiological solutions. The forces of interaction between an AFM cantilever tip and a uniform lawn of bacteria immobilized on glass were determined. By comparing the interactions of cantilever tips with lawns of isogenic E. coli strains carrying genetic lesions that alter their cell surface composition, it was possible to evaluate the effect of macromolecules such as lipopolysaccharide and capsular polysaccharide on the adhesion process. Mutations that result in the synthesis of truncated lipopolysaccharide or in the overproduction of the negatively charged capsular polysaccharide colanic acid render the interaction of the bacteria with the AFM tip unfavorable due to increased electrostatic repulsion. Furthermore, AFM could be used to evaluate the adhesion of bacteria onto commercially relevant biomaterials. In one approach, micron-size polystyrene beads were attached to AFM tips which were then used to measure forces. Unfortunately, this approach is limited by the meager number of materials manufactured as beads of a size suitable for AFM measurements. As an alternative approach, AFM cantilever tips were coated with a confluent layer of bacteria and used to probe planar surfaces. In this configuration, AFM could be employed to measure the force of interaction between virtually any bacterium and surface of interest.

Bacterial Adhesion↗

PubMed1998

Valve replacement under retrograde warm-blood cardioplegia. Results in 287 patients.

We studied 287 consecutive patients who underwent valve replacement procedures under retrograde warm-blood cardioplegia between 1 March 1992 and 30 June 1997 (64 months). Some of the procedures were performed in combination with other operations (70), but most (217) were isolated. Thirty patients had undergone previous "open" procedures and another 25 patients had undergone prior "closed" procedures. The 30-day postoperative mortality rate was 3.8% (11 deaths). In 7 patients, the cause of death was not cardiogenic. We did not observe any instance of right ventricular failure, perforation of the coronary sinus, phrenic nerve palsy, or wound infection. These results indicate that retrograde warm-blood cardioplegia provides excellent myocardial protection of both ventricles during valve replacement.

Adult↗

PubMed1997

Karyotypic abnormalities in fibroadenomas of the breast.

Short-term cultures of 50 fibroadenomas of the breast were cytogenetically analyzed. Nine tumors were found to display clonal chromosome aberrations. One had multiple, cytogenetically unrelated clones, whereas the others had a single abnormal clone each. Four cases had one balanced translocation as the sole anomaly, and one had a complex intrachromosomal rearrangement of chromosome 3, leading to loss of 3p material. One fibroadenoma had a single numerical aberration, and one had supernumerary ring chromosomes. The remaining 2 cases had both numerical and structural aberrations. The only recurrent alterations were trisomy 20 and rearrangement of chromosome arm 1p. The finding of similar chromosomal aberrations in fibroadenomas and carcinomas suggests that women with karyotypically abnormal fibroadenomas may have an increased risk of developing subsequent breast cancer. If so, different chromosome anomalies might have different pathogenetic and/or prognostic significance.

Adolescent↗

PubMed1997

In vitro scanning saturation mutagenesis of an antibody binding pocket.

We have combined PCR mutagenesis with in vitro transcription/translation and ELISA for the rapid generation and characterization of antibody mutants. The PCR products are used directly as the template for the in vitro transcription/translation reactions and because no cloning steps are required, the in vitro saturation mutagenesis of one residue can be completed in duplicate within a week by a single investigator. In vitro scanning saturation mutagenesis was used to analyze the role and plasticity of six key contact residues (H:Tyr-33, H:Asn-35, H:Tyr-50, H:Trp-100, L:Val-94, and L:Pro-96) in the binding pocket of a single chain Fv antibody derived from the 26-10 monoclonal antibody. A total of 114 mutant antibodies were produced; all 19 substitutions at each of the 6 chosen positions. The mutants were analyzed for binding to digoxin, digitoxin, digoxigenin, and ouabain resulting in the generation of a comprehensive data base of 456 relative affinity values. Excellent agreement between the relative affinity values obtained with in vitro synthesized mutant antibodies and equilibrium affinity data obtained with previously reported purified mutant monoclonal antibodies was observed. Approximately 75% of the single amino acid mutants exhibited significant binding to one or more of the digoxin analogs. Mutations that alter and, in some cases, reverse specificity for the different digoxin analogs were identified. In vitro scanning saturation mutagenesis represents a new tool for protein structure-function and engineering studies and can be interfaced with laboratory automation so that an even higher throughput of protein mutants can be constructed and analyzed.

Binding Sites, Antibody↗

PubMed1997

Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines.

In recent years there has been considerable progress towards the development of expression systems for the display of heterologous polypeptides and, to a lesser extent, oligosaccharides on the surface of bacteria or yeast. The availability of protein display vectors has in turn provided the impetus for a range of exciting technologies. Polypeptide libraries can be displayed in bacteria and screened by cell sorting techniques, thus simplifying the isolation of proteins with high affinity for ligands. Expression of antigens on the surface of nonvirulent microorganisms is an attractive approach to the development of high-efficacy recombinant live vaccines. Finally, cells displaying protein receptors or antibodies are of use for analytical applications and bioseparations.

Bacterial Proteins↗

PubMed1997

Reduction of the periplasmic disulfide bond isomerase, DsbC, occurs by passage of electrons from cytoplasmic thioredoxin.

The Escherichia coli periplasmic protein DsbC is active both in vivo and in vitro as a protein disulfide isomerase. For DsbC to attack incorrectly formed disulfide bonds in substrate proteins, its two active-site cysteines should be in the reduced form. Here we present evidence that, in wild-type cells, these two cysteines are reduced. Further, we show that a pathway involving the cytoplasmic proteins thioredoxin reductase and thioredoxin and the cytoplasmic membrane protein DsbD is responsible for the reduction of these cysteines. Thus, reducing potential is passed from cytoplasmic electron donors through the cytoplasmic membrane to DsbC. This pathway does not appear to utilize the cytoplasmic glutathione-glutaredoxin pathway. The redox state of the active-site cysteines of DsbC correlates quite closely with its ability to assist in the folding of proteins with multiple disulfide bonds. Analysis of the activity of mutant forms of DsbC in which either or both of these cysteines have been altered further supports the role of DsbC as a disulfide bond isomerase.

Aprotinin↗

PubMed1996

Eukaryotic protein disulfide isomerase complements Escherichia coli dsbA mutants and increases the yield of a heterologous secreted protein with disulfide bonds.

Eukaryotic protein disulfide isomerase (PDI) is a 55-kDa enzyme with cysteine oxidoreductase, chaperone, and antichaperone activities that catalyzes disulfide formation and rearrangement in the eukaryotic endoplasmic reticulum. In Gram-negative bacteria, the formation of disulfide bonds in the periplasm is mediated by DsbA, a strong cysteine oxidase but an inefficient catalyst of disulfide bond isomerization with no known chaperone activity. We show that rat PDI (rPDI) secreted in the periplasmic space of Escherichia coli can catalyze the formation of disulfide bonds and complement several of the phenotypes of dsbA mutants. The function of rPDI was dependent on the dsbB gene, suggesting that the reoxidation of this eukaryotic enzyme involves direct interactions with bacterial redox proteins. Co-expression of rPDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) severalfold, an effect that was enhanced when reduced glutathione was added to the growth medium. Whereas PDI is thought to function primarily as an isomerase in the eukaryotic endoplasmic reticulum, rPDI failed to decrease the accumulation of two-disulfide folding intermediates of BPTI and thus did not appear to appreciably catalyze the rate-limiting step in the oxidative folding pathway of BPTI. These results demonstrate that expression of eukaryotic foldases in E. coli can be exploited to better understand their function in vivo and also to increase the yield of biotechnologically valuable proteins.

Animals↗

PubMed1996

Characterization of Escherichia coli expressing an Lpp'OmpA(46-159)-PhoA fusion protein localized in the outer membrane.

The Lpp'OmpA(46-159) hybrid protein can serve as an efficient targeting vehicle for localizing a variety of procaryotic and eucaryotic soluble proteins onto the E. coli surface, thus providing a system for several possible biotechnology applications. Here we show that fusion between Lpp'OmpA(46-159) and bacterial alkaline phosphatase (PhoA), a normally periplasmic dimeric enzyme, are also targeted to the outer membrane. However, protease accessibility experiments and immunoelectron microscopy revealed that, unlike other periplasmic proteins, the PhoA domain of these fusions is not exposed on the cell surface in cells having an intact outer membrane. Conditions that affect the formation of disulfide bonds and the folding of the PhoA domain in the periplasm not only did not facilitate targeting to the cell surface but led to lethality when the fusion was expressed from a high-copy-number plasmid. Furthermore, E. coli expressing the Lpp'OmpA(46-159)-PhoA fusion exhibited strain- and temperature-dependent alterations in outer-membrane permeability. Our results are consistent with previous studies with other vehicles indicating that PhoA is not displayed on the surface when fused to cell-surface expression vectors. Presumably, the enzyme rapidly assumes a tightly folded dimeric conformation that cannot be transported across the outer membrane. The large size and quaternary structure of PhoA may define a limitation of the Lpp'OmpA(46-159) fusion system for the display of periplasmic proteins on the cell surface. Alkaline phosphatase is a unique protein among a group of five periplasmic proteins (beta-lactamase, alkaline phosphatase, Cex cellulase Cex cellulose-binding domain, and a single-chain Fv antibody fragment), which have been tested as passengers for the Lpp'OmpA(46-159) expression system to date, since it was the only protein not displayed on the surface.

Alkaline Phosphatase↗

PubMed1996

Expression of correctly folded proteins in Escherichia coli.

Many heterologous polypeptides fail to fold into their native state when expressed in Escherichia coli; instead, they are either degraded by the cellular proteolytic machinery or accumulate in insoluble form, typically as inclusion bodies. Misfolding is a particularly vexing problem in the expression of mammalian proteins, especially those that are composed of multiple subunits, have several disulfide bonds, or contain prosthetic groups. Fortunately, bacteria exhibit a remarkable physiological plasticity that can be successfully exploited to improve protein folding. Significant yields of active heterologous proteins have been obtained through strategies that include the co-expression of homologous or heterologous folding accessory proteins, the optimization of growth conditions, and the use of fusion proteins. A flood of recent reports documenting the successful production of complex eukaryotic proteins in active form have demonstrated that bacteria can provide the proper environment for the folding of the vast majority of recombinant polypeptides.

Bacterial Proteins↗

PubMed1996

Expression of fos-like immunoreactivity in the hypothalamus of the ring dove (Streptopelia risoria) at the onset of incubation.

Fos immunocytochemistry was used in the ring dove (Streptopelia risoria) to identify areas in the forebrain and hypothalamus which might be involved in the expression of incubation behaviour and the associated changes in neuroendocrine function. In this species, both sexes participate in incubation which becomes fully established after laying the second egg in the two egg clutch. The eggs hatch after 14-15 days incubation. Observations were made in both sexes 16-18 h after laying the first egg, 3-5 h after laying the second egg, and after 12-14 days of incubation. Non-breeding birds were used as controls. Fos-labelled cells were observed in several forebrain areas in both sexes irrespective of reproductive status but their numbers did not change during incubation. In the hypothalamus, very few fos-labelled cells were seen in non-breeding birds but appeared in both sexes in the nucleus preopticus medialis after egg laying and after 12-14 days of incubation. Fos-labelled cells also appeared in the nucleus tuberis (=arcuate nucleus) in both sexes after laying the first or second egg and less abundantly, after 12-14 days incubation. Additionally, in males only, fos-labelled cells were seen after the laying of the first and second eggs in the rostral area of the nucleus tuberis close to the walls of the third ventricle. These observations suggest that the fos-labelled cells in the nucleus preopticus medialis may be involved in the expression of incubation behaviour while those in the nucleus tuberis may be involved in the regulation of gonadotrophin or prolactin secretion.

Animals↗

PubMed1996

Display of beta-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-beta-lactamase fusions.

Bacterial cell-surface exposure of foreign peptides and soluble proteins has been achieved recently by employing a fusion protein methodology. An Lpp'-OmpA(46-159)-Bla fusion protein has been shown previously to display the normally periplasmic enzyme beta-lactamase (Bla) on the cell surface of the Gram-negative bacterium Escherichia coli. Here, we have investigated the role of the OmpA domain of the tripartite fusion protein in the surface display of the passenger domain (Bla) and have characterized the effects of the fusion proteins on the integrity and permeability of the outer membrane. We show that in addition to OmpA(46-159), a second OmpA segment, consisting of amino acids 46-66, can also mediate the display of Bla on the cell surface. Other OmpA domains of various lengths (amino acids 46-84, 46-109, 46-128, 46-141 and 46-145) either anchored the Bla domain on the periplasmic face of the outer membrane or caused a major disruption of the outer membrane, allowing the penetration of antibodies into the cell. Detergent and antibiotic sensitivity and periplasmic leakage assays showed that changes in the permeability of the outer membrane are an unavoidable consequence of displaying a large periplasmic protein on the surface of E. coli. This is the first systematic report on the effects that cell surface engineering may have on the integrity and permeability properties of bacterial outer membranes.

Amino Acid Sequence↗