Fast Magnetic Field-Enhanced Linear Colloidal Agglutination Immunoassay.

We present the principle of a fast magnetic field enhanced colloidal agglutination assay, which is based on the acceleration of the recognition rate between ligands and receptors induced by magnetic forces. By applying a homogeneous magnetic field of 20 mT for only 7 s, we detect CRP (C-reactive protein) in human serum at a concentration as low as 1 pM for a total cycle time of about 1 min in a prototype analyzer. Such a short measurement time does not impair the performances of the assay when compared to longer experiments. The concentration range dynamic is shown to cover 3 orders of magnitude. An analytical model of agglutination is also successfully fitting our data obtained with a short magnetic pulse.

Cleavage-sensing redox peptide monolayers for the rapid measurement of the proteolytic activity of trypsin and alpha-thrombin enzymes.

Ferrocene (Fc)-labeled peptides are end-grafted onto gold electrodes via a flexible polyethylene glycol (PEG) linker, and their ability to act as substrates for proteolytic enzymes trypsin and alpha-thrombin is investigated by cyclic voltammetry. It is shown that whereas a short Fc-tetrapeptide substrate is rapidly cleaved by trypsin, a longer Fc-heptapeptide substrate is required for alpha-thrombin detection. However, in both cases it is observed that not all of the Fc-peptide chains present on the electrode surface are cleavable by the proteases and that the cleavage yield is actually controlled by the surface coverage in the Fc-peptide. Surface dilution of the Fc-peptide using a backfilling molecule such as MCH (6-mercapto-1-hexanol) was required to obtain a cleavage yield larger than 80%. The kinetics of Fc-peptide cleavage by trypsin or alpha-thrombin is then shown to be adequately described by Michaelis Menten kinetics, allowing enzymatic constants k(cat) and K(M) to be determined. The obtained rate constant values showed that the affinity of the enzymes for their respective Fc-peptide substrates is very high (i.e., low K(M) values) whereas that for the cleavage step itself is relatively low (low k(cat) values). Partial compensation of these parameters yields a fast response of the Fc-peptide electrodes to the proteases in solution in the 1-1000 nM range. The type of molecule used to backfill the Fc-peptide layers, either MCH or PEG(6) chains, is shown to modulate the activity of the proteases versus the Fc-peptide layers: in particular, the PEG(6) diluent is specifically shown to decrease the ability of alpha-thrombin to cleave its Fc-peptide substrate whereas trypsin activity is unaffected by the presence of PEG chains.

Total biosynthesis of hydrocortisone from a simple carbon source in yeast.

We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.

Interfering lipoproteins in magnetic field-assisted agglutination of superparamagnetic particles immunoassay.

OBJECTIVE: The technology of magnetic field-assisted immuno-agglutination of superparamagnetic particles allows sensitive detection of biomarkers in whole blood. However, we observed non-specific agglutination (NSA), due to interfering plasma proteins, that negatively affects C-reactive protein immunoassay. The objective of the study was to identify the plasma proteins involved and to eliminate these interferences. DESIGN AND METHODS: Plasma was fractionated by size exclusion HPLC and each fraction was tested for non-specific agglutination. In addition, plasma proteins bound to magnetic particles were analyzed by SDS-gel electrophoresis and identified by mass spectrometry. RESULTS: We found that NSA was due to the binding of some lipoproteins to the particles. NSA was observed in the presence of purified LDL and VLDL but not HDL. NSA was mediated by the binding of ApoB100 to magnetic particles through its heparin binding sites. These interferences could be eliminated by addition of heparin or other polyanions like dextran sulfate to the assay buffer. CONCLUSION: NSA results from the binding of some plasma lipoproteins to magnetic particles. The use of a polyanion to eliminate these interferences allows the formulation of a stable reagent.

Identification of the glycosylation site of the adenovirus type 5 fiber protein.

The fiber protein purified from the pool of nonincorporated viral protein after infection of cells with adenovirus type 5 exists as two forms separable by reverse-phase HPLC. As determined by mass spectrometry, this heterogeneity results from a difference in one O-linked N-acetylglucosamine (GlcNac). A western blot analysis using a monoclonal antibody directed against the GlcNac motif showed that only one of the two forms reacted with the antibody, suggesting that one form carries a single GlcNac and the other form has none. The ratio of glycosylated to nonglycosylated forms of fiber, which is about 1, is conserved in assembled viruses. After digestion of glycosylated fiber with endoproteinase GluC, isolation of the glycosylated peptide by reverse-phase HPLC, and chemical derivatization using dimethylamine, the site of glycosylation was located in the fiber shaft at serine 109 by mass spectrometry. Elimination of glycosylation by site-directed mutagenesis of fiber should help to understand the function of this postranslational modification.

Dehydroepiandrosterone (DHEA) metabolism in Saccharomyces cerevisiae expressing mammalian steroid hydroxylase CYP7B: Ayr1p and Fox2p display 17beta-hydroxysteroid dehydrogenase activity.

We have engineered recombinant yeast to perform stereospecific hydroxylation of dehydroepiandrosterone (DHEA). This mammalian pro-hormone promotes brain and immune function; hydroxylation at the 7alpha position by P450 CYP7B is the major pathway of metabolic activation. We have sought to activate DHEA via yeast expression of rat CYP7B enzyme. Saccharomyces cerevisiae was found to metabolize DHEA by 3beta-acetylation; this was abolished by mutation at atf2. DHEA was also toxic, blocking tryptophan (trp) uptake: prototrophic strains were DHEA-resistant. In TRP(+) atf2 strains DHEA was then converted to androstene-3beta,17beta-diol (A/enediol) by an endogenous 17beta-hydroxysteroid dehydrogenase (17betaHSD). Seven yeast polypeptides similar to human 17betaHSDs were identified: when expressed in yeast, only AYR1 (1-acyl dihydroxyacetone phosphate reductase) increased A/enediol accumulation, while the hydroxyacyl-CoA dehydrogenase Fox2p, highly homologous to human 17betaHSD4, oxidized A/enediol to DHEA. The presence of endogenous yeast enzymes metabolizing steroids may relate to fungal pathogenesis. Disruption of AYR1 eliminated reductive 17betaHSD activity, and expression of CYP7B on the combination background (atf2, ayr1, TRP(+)) permitted efficient (>98%) bioconversion of DHEA to 7alpha-hydroxyDHEA, a product of potential medical utility.