Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media. IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E. coli become adsorbed, in addition to our target recombinant protein, on these IMAC supports via multipoint weak adsorptions; (b) the multimeric poly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits. In this way, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole). The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are now adsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole. In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports). Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface. However, the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface.     

On macroscopic description of photorefractive phenomena

A phenomenological approach, in which the current density is related in some functional manner to the electric field and to the light intensity, is developed. The validity and advantages of this macroscopic formulation are discussed by comparing its predictions with those derived from the particular microscopic models. Finally, some general phenomenological equations are presented which include, among others, photon drag and the magneto-photovoltaic effect. Received: 6 November 1998 / Revised version: 15 January 1999 / Published online: 12 April 1999     

Nanomechanics of the formation of DNA self-assembled monolayers and hybridization on microcantilevers

Biomolecular interactions over the surface of a microcantilever can produce its bending motion via changes of the surface stress, which is referred to nanomechanical response. Here, we have studied the interaction forces responsible for the bending motion during the formation of a self-assembled monolayer of thiolated 27-mer single-stranded DNA on the gold-coated side of a microcantilever and during the subsequent hybridization with the complementary nucleic acid. The immobilization of the single-stranded DNA probe gives a mean surface stress of 25 mN/m and a mean bending of 23 nm for microcantilevers with a length and thickness of about 200 microm and 0.8 microm, respectively. The hybridization with the complementary sequence could not be inferred from the nanomechanical response. The nanomechanical response was compared with data from well-established techniques such as surface plasmon resonance and radiolabeling, to determine the surface coverage and study the intermolecular forces between neighboring DNA molecules anchored to the microcantilever surface. From both techniques, an immobilization surface density of 3 x 10(12) molecules/cm(2) and a hybridization efficiency of 40% were determined. More importantly, label-free hybridization was clearly detected in the same conditions with a conventional sensor based on surface plasmon resonance. The results imply that the nanomechanical signal during the immobilization process arises mainly from the covalent attachment to the gold surface, and the interchain interactions between neighboring DNA molecules are weak, producing an undetectable surface stress. We conclude that detection of nucleic acid hybridization with nanomechanical sensors requires reference cantilevers to remove nonspecific signals, more sensitive microcantilever geometries, and immobilization chemistries specially addressed to enhance the surface stress variations.     

Optical damage control via the Fe2+/Fe3+ ratio in proton-exchanged LiNbO3 waveguides

Intensity thresholds for the onset of optical damage in alpha-phase proton-exchanged waveguides on undoped LiNbO(3) have been increased from the substrate value to a factor 500 greater. This has been achieved by increasing the exchange time, which in turn increases the [Fe(2+)]/[Fe(3+)] ratio in the guide. Intensity thresholds have been measured with a single-beam configuration, while [Fe(2+)]/[Fe(3+)] ratios have been determined from decay measurements during optical erasure of photorefractive gratings. In heavily Fe-doped guides, thresholds are too small to be measured, but the dependence of the Fe(2+) absorption on the exchange time appears similar to undoped samples.     

Excitation and interrogation of whispering-gallery modes in optical microresonators using a single fused-tapered fiber tip

We show that whispering-gallery modes (WGMs) in optical microresonators can be excited and detected using a fused-tapered fiber tip (FTFT). The fabrication of FTFTs is simple and inexpensive; they are robust and allow the excitation and interrogation of the resonances with a single fiber. Excitation of high-Q WGMs in silica microcylinders and microspheres is demonstrated.