158 results about "Protein crystallization" patented technology

A method which can control crystallization of a biopolymer such as protein is provided. A silicon crystal (15) whose valence electrons are controlled to be capable of controlling the concentration of holes or electrons of the surface part in response to the environment of a buffer solution (14) containing the biopolymer such as protein is brought into contact with the solution (14), for getting a crystal of the biopolymer deposited on the surface of the silicon crystal (15). Crystallization is controlled by an electrical state which is generated by the controlled valence electrons on the surface of the silicon crystal (15).

Methods of using protein crystallization droplet actuators are provided. Protein sample droplets and reagent droplets are dispensed, transported, and merged to yield an array of crystallization conditions by electrowetting droplet operations in a gap comprising oil filler fluid. The oil filler fluid is doped with a surfactant that enhances droplet operations using the protein sample.

Protein crystallization droplet actuators, systems and methods are provided. According to one embodiment, a droplet actuator for providing an array of crystallization conditions is provided and includes: (a) two or more processing reservoirs; (b) two or more dispensing reservoirs collectively comprising two or more crystallization reagents; and (c) a port for introducing a sample for crystallization analysis. Systems including the droplet actuator, methods of providing an array of crystallization conditions, and methods of identifying crystallization conditions are also provided.

The use of microfluidic structures enables high throughput screening of protein crystallization. In one embodiment, an integrated combinatoric mixing chip allows for precise metering of reagents to rapidly create a large number of potential crystallization conditions, with possible crystal formations observed on chip. In an alternative embodiment, the microfluidic structures may be utilized to explore phase space conditions of a particular protein crystallizing agent combination, thereby identifying promising conditions and allowing for subsequent focused attempts to obtain crystal growth.

Embodiments of the present invention relate to methods and apparatuses for the discretization and manipulation of sample volumes that is simple, robust, and versatile. It is a fluidic device that partitions a sample by exploiting the interplay between fluidic forces, interfacial tension, channel geometry, and the final stability of the formed droplet and / or discretized volume. These compartmentalized volumes allow for isolation of samples and partitioning into a localized array that can subsequently be manipulated and analyzed. The isolation of the discretized volumes along with the device's inherent portability render our invention versatile for use in many areas, including but not limited to PCR, digital PCR, biological assays for diagnostics and prognostics, cancer diagnosis and prognosis, high throughput screening, single molecule and single cell reactions or assays, the study crystallization and other statistical processes, protein crystallization, drug screening, environmental testing, and the coupling to a wide range of analytical detection techniques for biomedical assays and measurements. The minimal fluid interconnects and simple flow geometry makes the device easy to use and implement, economical to fabricate and operate, and robust in its operations.

An apparatus that automatically captures, stores and analyzes images of crystallization experiments contained in a number of crystallization plates. The apparatus includes a plate nest capable of accommodating protein crystallization plates of a plurality of different types, image acquisition optics, including an objective lens and an image capturing device, for focusing an image of a crystallization well, a light source including a bright field illumination device and a dark field illumination device, a nest positioning controller for moving the position of the plate nest with respect to the image acquisition optics to align various selected wells with said objective lens for imaging of the content of the wells. A database stores experiment information associated with each of the crystallization plates, the experiment information including identification of specific crystal forming parameter values, each of the crystallization plates is identified in said database by a unique identification code. A crystallization imaging controller controls crystallization imaging by retrieving the experiment information for each crystallization plate inserted into the apparatus, and controlling the nest positioning controller and the image acquisition optics in accordance with the retrieved experiment information. The apparatus captures multiple images of each crystal site using different light source and polarization conditions, and processes the multiple images to form extended fused images of each crystal site.

A static fluid and a second fluid are placed into contact along a microfluidic free interface and allowed to mix by diffusion without convective flow across the interface. In accordance with one embodiment of the present invention, the fluids are static and initially positioned on either side of a closed valve structure in a microfluidic channel having a width that is tightly constrained in at least one dimension. The valve is then opened, and no-slip layers at the sides of the microfluidic channel suppress convective mixing between the two fluids along the resulting interface. Applications for microfluidic free interfaces in accordance with embodiments of the present invention include, but are not limited to, protein crystallization studies, protein solubility studies, determination of properties of fluidics systems, and a variety of biological assays such as diffusive immunoassays, substrate turnover assays, and competitive binding assays.

A method and devices for rapid cooling of small biological samples by plunging them in a cryogenic liquid, such as liquid nitrogen, or contacting them with a cryogenic metal surface, reduce or eliminate the cold gas layer that forms above the liquid cryogens or cryogenic surfaces, producing an abrupt transition from ambient (e.g., room) temperature to the cryogen temperature as the sample enters the liquid or contacts the surface. To reduce or eliminate the effects of the cold gas layer, a flow of warm dry gas can be directed along the plunge path, for example. By removing this cold gas layer, cooling times for a 10 micron sample (the size of single cells and the smallest protein crystals now used protein crystallography) will decrease to ˜0.001 s.

Crystallization Photoresist (PR) apparatus and methods which allow for fast screening and determination of protein crystallization conditions with small protein quantities and rapid crystallization. The apparatus comprise a first region comprising a first nucleation catalyst material and a second region comprising a second nucleation catalyst material, with the first and second regions positioned adjacent to each other and configured to support at least one crystal, and with the first region having a variation in a nucleation property of the first nucleation catalyst material in the first region. The crystal may be supported at an interface of the adjacent regions. The methods comprise providing a first region of a first nucleation catalyst material and a second region of a second nucleation catalyst material adjacent said first region, with the first region having a variation in a nucleation property of the first nucleation catalyst material, exposing the first and second regions to a solution of a selected molecule, and growing at least one crystal of the molecule in association with the first and second regions.

The invention provides a semi-contact under-oil continuous droplet sample applying and liquid adding method, which is suitable for sequentially operating a droplet array system. According to the method, the distance between the pointed end of a capillary tube sample applying needle and the lower surface of a micro-hole (or a generated droplet) is controlled accurately, and the affinity or interface tension interaction between the droplet and the surface (or the generated droplet) is utilized, so that rapid and reliable continuous droplet sample applying or continuous liquid adding is realized, and the problem of cross infection during sample applying is solved effectively. The method is suitable for biochemical analysis screening researches such as high-flux medicament screening, protein crystallization condition screening, enzyme kinetics research and drug toxicity determination.

The present invention relates to methods and apparatus for promoting rapid formation of biomolecule crystals from a solution of biomolecules, preferably proteins, wherein the protein solution undergoes rapid concentration according to its isoelectric point in an electric field. Protein crystallization according to the methods of the present invention takes place within a period of hours or less.

Embodiments of the present invention relate to methods and apparatuses for the discretization and manipulation of sample volumes that is simple, robust, and versatile. It is a fluidic device that partitions a sample by exploiting the interplay between fluidic forces, interfacial tension, channel geometry, and the final stability of the formed droplet and / or discretized volume. These compartmentalized volumes allow for isolation of samples and partitioning into a localized array that can subsequently be manipulated and analyzed. The isolation of the discretized volumes along with the device's inherent portability render our invention versatile for use in many areas, including but not limited to PCR, digital PCR, biological assays for diagnostics and prognostics, cancer diagnosis and prognosis, high throughput screening, single molecule and single cell reactions or assays, the study crystallization and other statistical processes, protein crystallization, drug screening, environmental testing, and the coupling to a wide range of analytical detection techniques for biomedical assays and measurements. The minimal fluid interconnects and simple flow geometry makes the device easy to use and implement, economical to fabricate and operate, and robust in its operations.

Biomolecule arrays on a substrate are described which contain a plurality of biomolecules, such as coding nucleic acids and / or isolated polypeptides, at a plurality of discrete, isolated, locations. The arrays can be used, for example, in high throughput genomics and proteomics for specific uses including, but not limited molecular diagnostics for early detection, diagnosis, treatment, prognosis, monitoring clinical response, and protein crystallography.

The invention discloses a micro-fluidic chip for high-flux automatically rationing and mixing and a use method and an application of the chip. The micro-fluidic chip is a combined micro-fluidic chip and consists of a micro-fluidic chip main body and a pre-degassing PDMS (polydimethyl siloxane) pump body which are combined; and based on the chip, negative pressure is produced in a closed micro-pipe system to form a fluid driving force by using the high dissolution property of the PDMS pump body to gases after de-gassing treatment, simultaneously a capillary valve is constructed by using the chip micro-pipe surface property and combining the geometric design, and automatic filling, rationing and mixing of fluid in the micro-fluidic chip main body are achieved through the coordinate action of the negative pressure driving and the capillary valve. Finally, the invention discloses the micro-fluidic chip used for high-flux screening of protein crystallization conditions.

The present invention relates to methods and apparatus for promoting rapid formation of biomolecule crystals from a solution of biomolecules, preferably proteins, wherein the protein solution undergoes rapid concentration according to its isoelectric point in an electric field. Protein crystallization according to the methods of the present invention takes place within a period of hours or less.

The invention discloses a gradient micro-droplet array forming method based on sequential injection and microfluidic technology. The gradient micro-droplet array forming method comprises following steps: step 1, one of a diluent and a sample I is taken using a capillary tube; step 2, the other one of the diluent and the sample I is taken using the capillary tube, and direct contact of the diluent with the sample I is ensured so as to form a sample zone belt with axial concentration gradient; and step 3, the sample zone belt is injected into certain areas of a microporous array chip via the capillary tube so as to obtain sample droplet array with different concentration on the microporous array chip. Advantages of the gradient micro-droplet array forming method are that: concentration gradient generation throughput is high, concentration information is abundant, automatic degree is high; sample consumption is low, and devices and principles are simple. The gradient micro-droplet array forming method can be used for biochemical analysis and screening such as high throughput drug screening, compound toxicity determination, protein crystallization condition screening, catalyst screening, and enzyme dynamic analysis.

The invention relates to an expression, purification, crystallization method, structure elucidation and the field of application technology of forulic acid decarboxylase, which belongs to the field of molecular biology and microbiology application. The invention obtains the coding area sequence of the key enzyme forulic acid decarboxylase which decomposes forulic acid to generate 4-vinyl guaiacol in Enterobacter sp.Px6-4 by the gene clone method, expresses the enzyme in a large amount in the colon bacillus ( E.coli BL21 ) by the heterogenous expressing technology, obtains the forulic acid decarboxylase pure products by the purification method and the forulic acid decarboxylase protein crystallization by the hanging-drop crystallization process, and grasps the structure and active centre of the enzyme. The invention can obtain a large amount of highly active forulic acid decarboxylase, and provide theoretical guidance for reconstructing engineering bacteria and improving the output of 4-vinyl guaiacol by analyzing the active centre of the enzyme, thus having good potential application.

The present disclosure provides an effective method for the crystallization of proteins at high pressure. A preferential excluding agent, and optionally other reagents may be incorporated into the method. The method is applicable to substantially all proteins.

The invention discloses a zwitterion-containing water-soluble cross-linking agent as well as a preparation method and application thereof. The structural formula of the compound is a formula I. The compound has good water solubility and anti-nonspecific adsorption feature, so that the compound can be used as a cross-linking agent to prepare the polymer with high cross-linking degree, the obtained polymer has excellent biological pollution resistant feature, and can be widely applied to the fields such as biomedicine and materials. The water-soluble cross-linking agent is capable of reducing the protein aggregation speed, so that the protein molecule can be more easily presented in crystal form, and therefore the water-soluble cross-linking agent can be used for synthesizing the biomaterial for promoting protein crystallization. Furthermore, the cross-linking agent molecule comprises two-NH-C=O groups capable of generating hydrogen bond interaction with the carbohydrate (-OH) abound on the surface of microorganism; therefore, the cross-linking agent molecule can be used as an additive to prepare a molecular imprinting film material so as to selectively separate the microorganism (structural formula).

The present invention relates to a method of precipitating protein crystals from a protein-containing sample. The present invention also relates to a novel microarray and a novel device for screening for protein crystallization condition. Furthermore, the present invention relates to a method of conveniently and quickly screening for protein crystallization conditions using the microarray or the device having highly integrated and held crystallization conditions even with an extremely small quantity of a sample.

An automated biological sample analysis system and method for use in incubating and analyzing multiple samples for protein crystallization. A temperature controlled cabinet houses sample storage, sample transport, and sample imaging systems. The operation of the system is automated and can be controlled by software, which can be reconfigured remotely. An array of storage shelves includes multiple shelf columns arranged around a core. Multiple banks of removable shelves arranged as magazines are accessed through a door on the cabinet. Each shelf stores a multi-well plate and different sizes can be stored in different shelves. The core houses a sample transport system that includes a multi-axis robot that rotates about a vertical axis to access the shelves in the shelf array. The transport system retrieves and replaces the multi-well plates in the shelves and can move plates from the shelves to an imaging system where each sample can be automatically imaged.

A high-density high-throughput microplate and methods for simultaneously screening a plurality of protein crystallization solutions and for producing diffraction quality protein crystals in a vapor-diffusion environment are disclosed. The microplate has defined side-by-side paired chambers of equal size, wherein the side-by-side paired chambers have a maximum volume of about 8 μl, and wherein the paired chambers have a vapor channel, therein providing vapor exchange between the side-by-side paired chambers. The microplate further includes a membrane to seal the surface of the microplate. The microplate is adapted to receive a crystallization solution in one of the side-by-side paired chambers and a protein solution in the other of the side-by-side paired chambers, wherein the protein solution and the crystallization solution interact via a vapor diffusion process, which enables the formation of protein crystals within the chamber that contains the protein solution.

The use of microfluidic structures enables high throughput screening of protein crystallization. In one embodiment, an integrated combinatoric mixing chip allows for precise metering of reagents to rapidly create a large number of potential crystallization conditions, with possible crystal formations observed on chip. In an alternative embodiment, the microfluidic structures may be utilized to explore phase space conditions of a particular protein crystallizing agent combination, thereby identifying promising conditions and allowing for subsequent focused attempts to obtain crystal growth.

The present invention relates to a process for producing high-quality crystals of protein or organic substances easily and efficiently. A solution of protein or an organic substance is prepared and then is cooled slowly to be supersaturated to a low degree. This supersaturated solution is irradiated with a femtosecond laser 10. A local explosion phenomenon occurs at the focal point of the laser and thereby a crystalline nucleus is generated. A high-quality crystal is obtained when a crystal is grown on the crystalline nucleus over a long period of time. The femtosecond laser to be used herein can be a titanium:sapphire laser having a wavelength of 800 nm, a duration of 120 fs, a frequency of 1 kHz, and an output of 400 mW.

This invention provides a method for crystallizing proteins from protein solution with ionic liquid-water mixed solution. The ionic liquid-water mixed solution is composed of ionic liquid 1-85wt.%, and buffer solution 1-25 wt.%, and the pH value is 4.0-11.0. The method has a wide process conditions, and can obtain protein single crystals with different morphologies under different conditions. The obtained protein single crystals have such advantages as high diffractive intensity, high repeatability and high operability.

The invention discloses a protein crystallization screening 96 condition kit using polyethylene glycol as a precipitator and aims to solve the technical problem existing in the conventional 96 condition screening kit that the success rate of protein crystallization condition screening is low. The PEG adopted in the technical scheme is the top eight types PEG which are most frequently used according to the statistical data of a BMCD database rather than selected randomly, so that the crystallization success rate of biological macromolecules is obviously improved. For example, the success rate of canavaline is increased from 4.2 percent in background technology to 81.2 percent, the success rate of lysozyme is increased from 29.2 percent in background technology to 46.9 percent, the success rate of protease K is increased from 58.3 percent in background technology to 66.7 percent, and the success rate of catalase is increased from 15.6 percent in background technology to 30.2 percent. In addition, the kit uses the PEG as the precipitator, a few types of reagents are used, and compared with the background technology kit, the kit of the invention is lower in cost.

Herein is described and optimum solubility screen in which a panel of buffers and many additives are provided in order to obtain the most homogeneous and monodisperse protein condition for protein crystallization. The present methods are useful for proteins that aggregate and cannot be concentrated prior to setting up crystallization screens. A broad range of buffers is intended for use in this screen. A high-throughput method using the hanging-drop method and vapor diffusion equilibrium and a panel of twenty-four buffers is further provided. After monitoring precipitation, the conditions leading to clear drops are selected for evaluation, preferably dynamic light scattering (DLS) characterization. If the DLS results are not optimal, a series of additives are tested in the presence of the best buffer selected from the initial screen and again DLS is used to determine the best condition. Using the present methods, 14 poorly behaving proteins have been screened, resulting in 11 of the proteins having highly improved DLS results allowing concentration of the proteins, and 9 were crystallized.

The present invention provides a method for selecting a buffer solution for protein crystallization. The method comprises: preparing a uniformly distributed buffer solution to dissolve protein; mixing a crystallization reagent and the protein solution or mixing a crystallization reagent, the buffer solution and a protein sample to prepare a protein crystallization solution; and standing in a sealed crystallization plate until crystallization. According to the present invention, the appropriate protein crystallization buffer solution pH value condition is selected, such that protein solubility is changed in a wide range, and protein crystal nucleation probability is increased so as to increase crystallization efficiency.