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Biomedical subjects

A E Cass

Publications and source records attributed to A E Cass.

At least 19 recordsLinked to original sources
PubMed2001

Introduction of a (poly)histidine tag in L-lactate dehydrogenase produces a mixture of active and inactive molecules.

A (poly)histidine tag was fused to either the N- or the C-terminus of L-lactate dehydrogenase (LDH) of Bacillus stearothermophilus to facilitate purification and immobilization of these enzymes. The C-terminally tagged enzyme displayed lower activity compared both to the wild-type and to the N-terminally tagged variant. The reason for this loss of activity was investigated by affinity chromatography of the enzymes on a 5'-AMP-Sepharose resin and by size-exclusion chromatography. The C-terminally tagged enzyme could be separated into an inactive, unbound fraction and an active, bound fraction. Further differences between the C-terminally tagged enzyme and the N-terminally tagged and wild-type LDH were observed on size-exclusion chromatography of the three enzymes. These data suggest that the introduction of a "his-tag" at the C-terminus may induce misfolding of the LDH and serve as a warning that the introduction of a (poly)histidine tag can produce unforseen changes in a protein.

Chromatography, Affinity↗
PubMed2001

A factorial analysis of silanization conditions for the immobilization of oligonucleotides on glass surfaces.

The modification of glass surfaces with (3-mercaptopropyl)trimethoxysilane and the application of this to DNA chip technology are described. A range of factors influencing the silanization method, and hence the number of surface-bound, chemically active thiol groups, were investigated using a design of experiment approach based on analysis of variance. The number of thiol groups introduced on glass substrates were measured directly using a specific radiolabel, [14C]cysteamine hydrochloride. For liquid-phase silanization, the number of surface-bound thiol groups was found to be dependent on both postsilanization thermal curing and silanization time and relatively independent of silane concentration, reaction temperature, and sample pretreatment. Depending on the conditions used in liquid-phase silanization, (1.3-9.0) x 10(12) thiol groups/cm2 on the glass samples were bound. The reliability and repeatability of liquid- and vacuum-phase silanization were also investigated. Eighteen-base oligonucleotide probes were covalently attached to the modified surfaces via a 3'-amino modification on the DNA and subsequent reaction with the cross-linking reagent N-(gamma-maleimidobutyryloxy) succinimide ester (GMBS). The resulting probe levels were determined and found to be stoichiometric with that of the introduced thiol groups. These results demonstrate that silanization of glass surfaces under specific conditions, prior to probe attachment, is of great importance in the development of DNA chips that use the simple concept of the covalent attachment of presynthesized oligonucleotides to silicon oxide surfaces.

Factor Analysis, Statistical↗
PubMed2001

[Cloning and expression of Aspergillus niger glucose oxidase gene in methylotrophic yeast].

The DNA fragment encoding A. niger glucose oxidase was amplified by PCR using A. niger genomic DNA as template, and was cloned into vector of pPIC9 for expression in Pichia pastoris. When transformed into methylotrophic yeast Pichia pastoris GS115, The constructed plasmid pPICGOD1 directed the synthesis and secretion of functionally active GOD. After induction in MM medium for 4 days, the GOD activity in the medium reached 30-40 u/mL. SDS-PAGE revealed that recombinant yeast GOD was expressed up to 60%-70% of the total soluble protein, and the secreted GOD could be purified to electrophoretic homogeneity with one purification step using Q Sepharose Fast Flow ion exchange chromatography. The recombinant yeast GOD had very high catalytic activity, showed about 1.6-fold increase of specific activity over the commercial A. niger GOD. Kinetic analysis clearly demonstrated that recombinant yeast GOD showed similar substrate affinity for glucose to A. niger GOD, but the turnover number of the GOD from yeast was determined to be much higher than that of A. niger GOD. In addition, the linear range of glucose electrode made with recombinant yeast GOD was efficiently widened due to the high catalytic activity of yeast GOD.

Aspergillus niger↗
PubMed2000

Protein adsorption on nanoporous TiO2 films: a novel approach to studying photoinduced protein/electrode transfer reactions.

We have investigated the use of nanoporous TiO2 films as substrates for protein immobilisation. Such films are of interest due to their high surface area, optical transparency, electrochemical activity and ease of fabrication. These films moreover allow detailed spectroscopic study of protein/electrode electron transfer processes. We find that protein immobilisation on such films may be readily achieved from aqueous solutions at 4 degrees C with a high binding stability and no detectable protein denaturation. The nanoporous structure of the film greatly enhances the active surface area available for protein binding (by a factor of up to 850 for an 8 microns thick film). We demonstrate that the redox state of proteins such as immobilised cytochrome-c (Cyt-c) and haemoglobin (Hb) may be modulated by the application of an electrical bias potential to the TiO2 film, without the addition of electron transfer mediators. The binding of Cyt-c on the TiO2 films is investigated as a function of film thickness, protein concentration, protein surface charge and ionic strength. We demonstrate the potential use of immobilised Hb on such TiO2 films for the detection of dissolved CO in aqueous solutions. We further show that protein/electrode electron transfer may be initiated by UV bandgap excitation of the TiO2 electrode. Both photooxidation and photoreduction of the immobilised proteins can be achieved. By employing pulsed UV laser excitation, the interfacial electron transfer kinetics can be monitored by transient optical spectroscopy, providing a novel probe of protein/electrode electron transfer kinetics. We conclude that nanoporous TiO2 films may be useful both for basic studies of protein/electrode interactions and for the development of novel bioanalytical devices such as biosensors.

Adsorption↗
PubMed1998

DNA optical sensor: a rapid method for the detection of DNA hybridization.

A DNA optical sensor system is proposed based on the combination of sandwich solution hybridization, magnetic bead capture, flow injection and chemiluminescence for rapid detection of DNA hybridization. Bacterial alkaline phosphatase (phoA) gene and Hepatitis B virus (HBV) DNA were used as target DNA. A biotinylated DNA probe was used to capture the target gene onto the streptavidin-coated magnetic beads and a calf intestine alkaline phosphatase (CAP)-labelled DNA probe was used for subsequent enzymatic chemiluminescence detection. The detection cycle was less than 30 min, excluding the DNA hybridization time, which was about 100 min. Both the phoA gene and HBV DNA could be detected at picogramme or femtomole level. No response signal was obtained when target DNA did not exist in the sample. Successive sample detection could be made by removing the magnetic field and a washing step.

Biosensing Techniques↗
PubMed1997

Mediated electrochemistry of peroxidases--effects of variations in protein and mediator structures.

The kinetics of a range of ferrocene derivatives with horseradish peroxidase (HRP), cytochrome c peroxidase (CCP) and 3 charge reversal mutants of cytochrome c peroxidase were measured using cyclic voltammetry. Substantial differences in rate constant (100 fold) were observed between HRP and CCP for the same mediator with smaller differences (4-5 fold) for different mediators with the same enzyme. The rate constant did not seem to be dependent on redox potential differences. Cluster analysis is proposed as a way of classifying mediator reactivity.

Cytochrome-c Peroxidase↗
PubMed1997

Purification and characterization of recombinant catalase-peroxidase, which confers isoniazid sensitivity in Mycobacterium tuberculosis.

The Mycobacterium tuberculosis katG gene encodes a dual-function enzyme called catalase-peroxidase, which confers sensitivity in M. tuberculosis to isonicotinic acid hydrazide. We have constructed a system for the high level expression of a recombinant form of this enzyme by amplifying the katG gene from the pYZ56 construct (1) and subcloning into a vector suitable for expression in Escherichia coli. The resulting plasmid, pTBCP, produced the catalase-peroxidase in large quantities, corresponding to 30% of total cell protein. The enzyme has been purified to homogeneity and appears to be a dimer in the native form. Using either hydrogen peroxide or t-butyl hydroperoxide and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) as substrates, kcat and Km values have been obtained for both catalatic and peroxidatic activities, respectively. The availability of significant quantities of an active, folded, recombinant form of M. tuberculosis catalase-peroxidase should thus facilitate future studies of its role in drug activation and antibiotic resistance.

Bacterial Proteins↗
PubMed1997

Alkaline phosphatase-Strep tag fusion protein binding to streptavidin: resonant mirror studies.

The properties of a fusion protein comprising a streptavidin recognition sequence (Strep tag) fused to the C terminus of Escherichia coli alkaline phosphatase are described. The catalytic properties were determined with p-nitrophenyl phosphate and compared to those of the native E. coli alkaline phosphatase. It was found that the Km values were similar in both cases (8 microM for transferase and 2 microM for hydrolase activities) whilst the Vmax values were lower for the fusion protein, possibly due to the presence of misfolded forms. An optical biosensor based on the resonant mirror was used to determine the binding kinetics between the fusion protein and the immobilised streptavidin. The association and dissociation rate constants were determined to be 2.1(+/-0.3) x 10(-2) microM(-1) s(-1) and 11(+/-0.2) x 10(-3) s(-1), respectively, which results in an equilibrium dissociation constant of 0.5 microM. This is larger than previously reported affinities based on titration calorimetry and may be a consequence of the presence of two streptavidin binding sequences on the dimeric alkaline phosphatase simultaneously binding to two subunits of streptavidin.

Alkaline Phosphatase↗
PubMed1997

Spectroscopic properties of an engineered maltose binding protein.

The maltose binding protein (MBP) has been site specifically labelled with a nitrobenzoxadiazole (NBD) group following mutation of a serine to a cysteine residue at position 337. The resulting protein shows a large ligand (maltose or beta-cyclodextrin) dependent increase in its steady-state fluorescence intensity. Analysis of the static (intensity and anisotropy) and dynamic (lifetime distributions) fluorescence of the NBD label as well as the tryptophan residues in both ligand-bound and ligand-free states of this molecule reveals complex multi-component decays that are interpreted in terms of a ligand-induced solvent shielding mechanism. In the context of the known crystal structures of the various forms of the maltose-binding protein (MBP), ligand-dependent changes in both the fluorescence parameters as well as the circular dichroism spectra of the NBD group are interpreted by a twisted intramolecular charge-transfer (TICT) mechanism, wherein ligand binding locks the NBD group into a conformation that prevents efficient relaxation of the excited state.

4-Chloro-7-nitrobenzofurazan↗
PubMed1994

Engineering the maltose binding protein for reagentless fluorescence sensing.

This paper describes a mutant of the maltose binding protein (MBP) in which the serine residue at position 337 is replaced by a cysteine residue using site-directed mutagenesis. The mutant MBP has an approximately 2-fold lower affinity for maltose, and the cysteine residue can be modified with 4-[N-(2-(iodoacetoxy)ethyl)-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD) and 6-acryloyl-2-(dimethylamino)-naphthalene (acrylodan). This combined genetic and chemical modification places the fluorophores close to the maltose binding site such that when the ligand is added the fluorescence intensity of the labels increases by 60-180% over that of the ligand-free form. This change is consistent with the fluorophores being buried when the conformation of the protein changes with maltose binding. Titration of the labeled mutant proteins yields dissociation constants for maltose of 62 +/- 0.2 and 0.8 +/- 0.01 microM respectively for the IANBD and acrylodan modifications. The application of this strategy of combined genetic and chemical modification to the development of reagentless fluorescence sensing is discussed.

2-Naphthylamine↗
PubMed1993

A step towards understanding the folding mechanism of horseradish peroxidase. Tryptophan fluorescence and circular dichroism equilibrium studies.

The guanidinium chloride denaturation/renaturation of the holo- and apo-horseradish peroxidase isoenzyme c (HRP) has been studied by fluorescence and circular dichroism spectroscopies. A distinct equilibrium intermediate of the apoprotein could be detected at low concentrations of guanidinium chloride (0.5 M). This intermediate has a secondary structure content like that of the native protein but a poorly defined tertiary structure. Renaturation of the apo-HRP is reversible and 100% activity could be obtained after addition of a twofold excess of free haem. The denaturation of the holo-HRP is more complex and occurs in two distinct steps; unfolding of the protein backbone and loss of the haem. The denatured protein folds back to its native conformation but the incorporation of the haem occurs only after the secondary structure is formed. Ca2+ appears to be important for the stability of the protein as the apo-HRP is more resistant to denaturation in the presence of Ca2+. The free-energy change during unfolding of the apo-HRP was determined in the absence and presence of Ca2+ and found to be 9.2 kJ/mol and 16.7 kJ/mol, respectively. The importance of Ca2+ to the protein stability was also supported by studies on the loss of the haem from the protoporphyrin-IX-apo-HRP complex.

Calcium↗
PubMed1992

Partition coefficient of luciferase from photobacteria in PEG/salt two aqueous phase system.

The relationship between the logarithmic partition coefficient (K) of luciferase of photobacteria and PEG MW in the PEG/salt two aqueous phase system was shown to be a linear function. The hydrophilic PEG of lower MW facilitated the partition of luciferase into the top PEG-riching phase and gave a higher K value, while PEG of higher MW, its hydrophobic characteristic, made more enzyme partition into the bottom salt-riching phase and lower K value was obtained. In the PEG/trivalent salt system, such as phosphate and citrate, there was a turning-point on the linear relation between the log K and the PEG MW, but which never appeared if a divalent salt such as sulfate, succinate or tartrate was used in the system. When the system was composed of homogeneous PEG and ammonium sulfate, the K value was increased with the increment of the salt concentration, but after the salt concentration had reached at certain level, the K value was uninfluenced. When two kinds of PEG with different MW were used in this system a minimal K value appeared at certain concentration of ammonium sulfate, and the K value was raised when the salt concentration was either increased or decreased. Neither the proportion of the two kinds of PEG nor their total concentration used in the system showed any effect on the above patterns, although the K value may give some corresponding changes. (ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Sulfate↗