35 results about "Kinase substrate" patented technology

The invention discloses a mycobacterium M. tuberculosis holoprotein chip and an application thereof. The protein chip has at least one of the following functions (1) to (7); the following 4,168 proteins are connected to the protein chip, and each protein independently forms a detection point. Experiments show that the mycobacterium M. tuberculosis holoprotein chip consists of a substrate and proteins containing more than 95 percent of mycobacterium M. tuberculosis genome codes. The holoprotein chip is obviously significant for looking for a serum biomarker, a substrate interacting with a micromolecular compound c-di-GMP, a kinase substrate interacting with protein kinase and a substrate interacting with macrophage. Compared with an existing research means, the holoprotein chip has global and high-throughput screening characteristics, research design and operation processes are simplified, and the research efficiency is remarkably improved.

In particular embodiments, the present invention provides methods of inhibiting smooth muscle cell responses and methods of treating or preventing vascular proliferative disease caused by smooth muscle cell migration and proliferation. More specifically, smooth muscle cell responses are inhibited by introducing an agent into smooth muscle cells, wherein the agent inhibits an activity of one or more members of the myristoylated alanine-rich C kinase substrate (MARCKS) family of proteins. The invention also provides methods of inhibiting expression of one or more genes encoding a member of the MARCKS family of proteins in a cell.

The invention provides methods for measuring the amount or activity of a component in a sample using a luminescent assay comprising a luciferase capable of generating light from a high-energy molecule. In one aspect, the invention provides methods for measuring the amount of NAD+ or the activity of an enzyme or enzyme series that results in the interconversion of NDA+ and NADH. In another aspect, the invention provides methods for measuring the amount of a kinase substrate, free inorganic phosphate, and phosphatase activity. In a further aspect, the invention provides methods for measuring the amount of cAMP in a sample.

A tyrosine kinase substrate is described. The tyrosine kinase substrate comprises a fusion protein which comprises a protein for labeling and a specific peptide fused with the protein for labeling. The specific peptide has an amino acid sequence including a glutamic acid residue and a tyrosine residue. The tyrosine kinase substrate is capable of being phosphorylated by a plurality of kinds of tyrosine kinases.

The present invention is concerned with method for pharmacologically profiling compounds using an array of substrates, in particular kinase substrates, immobilized on a porous matrix. This method was found particular useful in the prediction of drug response, i.e. to enable the distinction between responders and non-responders in the treatment of cells, tissues, organs or warm-blooded animals with the compound to be tested, and in compound differentiation.

The invention generally relates to compositions and methods for identifying and testing tyrosine kinase signaling pathway agonists and antagonists, and more particularly, methods and compositions for screening compounds and identifying compounds that will modulate the interaction of protein tyrosine kinase substrates with their intracellular ligands, as well as between their intracellular ligands and other members of the signaling pathway.

The disclosure relates to a cytoplasmic protein complex comprising: (a) a first recombinant fusion protein comprising a kinase, fused to a first interaction polypeptide; and (b) a second recombinant fusion protein comprising a domain comprising a reporter phosphorylation site, whereby the domain is fused to a second interaction polypeptide. The disclosure relates further to a method to detect compound-compound-interaction using the cytoplasmic protein complex, and to cells comprising such cytoplasmic protein complex.

The present invention relates to a biochip for the detection of phosphorylation and a method for measuring phosphorylation using the same, more precisely a biochip integrated with the substrate of kinase and a kit for measuring phosphorylation comprising the biochip and a radio-labeled co-factor, and a method for measuring phosphorylation using the same. The kit for the detection of phosphorylation of the present invention facilitates simple and fast measurement of phosphorylation with a minimum amount of a sample, compared with the conventional method using an antibody, because it uses a radioisotope. This chip and kit can be effectively used for the analysis of kinase activity since this method favors fast mass analysis.

The invention discloses a high flux screening method for screening an angiopoietin 2 receptor kinase inhibitor, which comprises the following steps: 1)establishment and optimization of a screening model of the angiopoietin 2 receptor kinase inhibitor: performing kinases concentration, incubation time, substrate concentration and ATP concentration experiments; 2)model reliability verification with positive drug: selecting kinases with appropriate concentration, ATP Km and substrate Km, respectively adding 2muml kinases and the substrate in each pore, adding 4muml positive drug in each pore according to concentration gradient, adding 2muml ATP for reacting, incubating at room temperature according to optimization time, adding 10muml stopping solution in each pore for stopping the reaction, incubating for 1 hour at room temperature and then detecting, analyzing data to obtain the positive drug IC50; and 3)verification of the high flux screen model: operating according to the above steps, using a Biomek NXP automation sampling apparatus and a Multidrop automatic liquid separator for feeding samples, and then calculating Z factors. The method has the advantages of simpleness and rapidity, high sensitivity, stable and reliable result and good reappearance, and can be used for high flux screening.

An in vitro protein kinase assay technology that (1) exhibits a high assay signal to background ratio (S / B) and range (S-B); (2) is homogenous; (3) is non-radioactive; and (4) does not require a phospho-specific antibody involves complexing a trivalent metal ion (e.g. Ga<3+>, Fe<3+>, Al<3+>, In<3+>, Ru<3+>, Sc<3+>, Y<3+>) to the surface of amplified luminescent proximity assay acceptor or donor beads, e.g., via a suitable linker such as nitrilotriacetic acid (NTA; also referred to as carboxymethyl-lysine), iminodiacetic acid (IDA), or an appropriately substituted N-containing heterocycle, for example a triazoheterocycle, for example a triazocyclononaneononane, such as 1-propylamino-4-acetato-1,4,7-triazacyclononane. A protein (or constituent part) or other kinase substrate is bound to the surface of the other of an amplified luminescent proximity assay acceptor or donor bead and, if phosphorylated, brought into proximity with the trivalent metal ion-complexed acceptor bead to generate a luminescent signal. Presence of a kinase inhibitor inhibits phosphorylation and therefore signal generation and, in this way, is detectable. As the invention described herein recognizes the presence or absence of phosphate groups on a protein, (or constituent part), or other biological macromolecule (e.g., mono, di, or trinucleotides, cyclic nucleotides or phosphate substituted inositols), it is broadly applicable to any phosphorlylation or dephosphorylation reaction enzymes and provides a highly robust and flexible assay format for protein kinases and other enzyme classes, including lipid kinases, phosphatases, phosphodiesterases and others.

The invention provides kinase substrates and methods comprising their use.

The invention discloses a high flux screening method for screening a tropomyosin-related kinase B inhibitor, which comprises the following steps: 1)establishment and optimization of a screening model of the tropomyosin-related kinase B inhibitor: performing kinases concentration, incubation time, substrate concentration and ATP concentration experiments; 2)model reliability verification with positive drug: selecting kinases with appropriate concentration, ATP Km and substrate Km, respectively adding 2muml kinases and the substrate in each pore, adding 4muml positive drug in each pore according to concentration gradient, adding 2muml ATP for reacting, incubating at room temperature according to optimization time, adding 10muml stopping solution in each pore for stopping the reaction, incubating for 1 hour at room temperature and then detecting, analyzing data to obtain the positive drug IC50; and 3)verification of the high flux screen model: operating according to the above steps, using a Biomek NXP automation sampling apparatus and a Multidrop automatic liquid separator for feeding samples, and then calculating Z factors. The method has the advantages of simpleness and rapidity, high sensitivity, stable and reliable result and good reappearance, and can be used for high flux screening.

The invention provides a cancer drug sensitivity prediction method and system, a storage medium and a terminal. The cancer drug sensitivity prediction method comprises the following steps: acquiring proteome and phosphorylated proteome data and drug sensitivity values of a plurality of patients; obtaining edge strength characteristics or node characteristic values of proteome and phosphorylated proteome data, and dividing the multiple drug effect sensitive values and the corresponding edge strength characteristics or node characteristic values into a training data set and a test data set; training an elastic network based on the training data set to predict cancer drug sensitivity, the elastic network being validated based on the test data set; and predicting the cancer drug sensitivity based on the trained elastic network. According to the cancer drug sensitivity prediction method and system, the storage medium and the terminal, drug effect discrimination of the targeted drug is realized by adopting the machine learning model based on the kinase substrate feature edge, so that accurate prediction of the cancer drug sensitivity is provided.

The invention relates to an agrobacterium strain for expressing recombined human albumin genes in plants, solving the problem that microorganisms and animals are not suitable for being used as reactors for industrial production of recombined human albumin currently. The recombined human albumin transgenic engineering strain is agrobacterium with recombined human albumin gene plasmids, and expressed protein is human albumin. According to the recombined human albumin gene sequence in the strain, a human albumin open-reading-frame basic-group 'basic sequence' of a Kozak sequence is inserted in front of a start codon, or a sequence (6xHis) of six repeated histidines is inserted in back of an N-end start codon on that basis, and an 'improved sequence' formed by sequence connection of code enterokinase substrates is arranged between the 6xHis and the albumin sequence. The recombined human albumin transgenic engineering strain can be applied to mass production of genetically recombined human albumin by transgenic plants.

Methods are provided to increase resistance to cell damage in a subject. The increase in resistance to cell damage in a subject in the subject is accomplished by decreasing activity of eEF2 kinase in the subject. The eEF2 kinase activity can be decreased by decreasing the amount of functional eEF2 kinase produced by the subject, including contacting the eEF2 kinase with a compound that inhibits phosphorylation of eEF2 kinase substrate or decreasing the amount of functional eEF2 kinase is decreased by reducing expression of a gene encoding the eEF2 kinase.

Methods are provided to increase resistance to cell damage in a subject. The increase in resistance to cell damage in a subject in the subject is accomplished by decreasing activity of eEF2 kinase in the subject. The eEF2 kinase activity can be decreased by decreasing the amount of functional eEF2 kinase produced by the subject, including contacting the eEF2 kinase with a compound that inhibits phosphorylation of eEF2 kinase substrate or decreasing the amount of functional eEF2 kinase is decreased by reducing expression of a gene encoding the eEF2 kinase.

The invention relates to a method for predicting phosphorylation site-specific kinase, which comprises the following specific steps of: constructing a kinase-substrate heterogeneous network according to data in a database, and calculating different weights given to edges of the kinase-substrate heterogeneous network; on the basis of the established network, predicting a potential kinase-substrate relationship by adopting a weighted maximum bipartite graph matching algorithm; for the queried phosphorylation site, intercepting an amino acid fragment of the queried phosphorylation site, and calculating sequence similarity scores between the fragment and substrate fragments catalyzed by different kinases; and predicting the catalytic kinase of the given phosphorylation site by combining the predicted kinase-substrate relationship and sequence similarity score. According to the prediction method of the phosphorylation site specific kinase, PPI network information and protein local sequence information are effectively combined; catalytic possibilities of each kinase under different thresholds are given according to the query site; compared with the existing method, the method has higher prediction accuracy on different kinases.

Disclosed are novel treatments for diseases and conditions caused by, directly or indirectly, high blood glucose levels increased gluconeogenesis. Such disease and conditions include, but are not limited to, type II diabetes, obesity, and cardiovascular conditions. Sam68, an RNA-binding adaptor protein and Src kinase substrate, is a novel regulator of hepatic gluconeogenesis and global and hepatic deletions of Sam68 significantly reduce blood glucose levels and the glucagon-induced expression of gluconeogenic genes. The treatments described herein may include inhibition of the activity of Sam68.

The present invention relates to apparatus and methods for separating and detecting enzyme substrates using separation gels. For example, the apparatus and methods can be used to separate and detect kinase substrates for further analysis. The apparatus and methods can also be used to detect enzyme inhibitors, such as kinase inhibitors.

Tyrosine kinase substrates are described herein that are phosphorylated by many and diverse tyrosine kinases, and are chemically stable relative to co-polymers of poly-EY or poly-EAY having random molecular weights in the range of 20-50 kDa. Tyrosine kinase substrate peptides are provided according to embodiments described herein which include an isolated tyrosine kinase substrate peptide having molecular weight in the range of about 0.5 kD-10 kD. Tyrosine kinase substrate peptides are provided according to embodiments described herein having no more than 50 amino acids. The peptides include 2-25 phosphorylation modules and each phosphorylation module has 2-3 amino acid residues.

The invention discloses a high flux screening method for screening an anaplastic lymphoma kinase inhibitor, which comprises the following steps: 1)establishment and optimization of a screening model of the anaplastic lymphoma kinase inhibitor: performing kinases concentration, incubation time, substrate concentration and ATP concentration experiments; 2)model reliability verification with positive drug: selecting kinases with appropriate concentration, ATP Km and substrate Km, respectively adding 2muml kinases and the substrate in each pore, adding 4muml positive drug in each pore according to concentration gradient, adding 2muml ATP for reacting, incubating at room temperature according to optimization time, adding 10muml stopping solution in each pore for stopping the reaction, incubating for 1 hour at room temperature and then detecting, analyzing data to obtain the positive drug IC50; and 3)verification of the high flux screen model: operating according to the above steps, using a Biomek NXP automation sampling apparatus and a Multidrop automatic liquid separator for feeding samples, and then calculating Z factors. The method has the advantages of simpleness and rapidity, high sensitivity, stable and reliable result and good reappearance, and can be used for high flux screening.

The invention provides an LRR (leucine-rich repeat) receptor kinase substrate searching method. The LRR receptor kinase substrate searching method includes: S1, incubating LRR receptor kinase along with ATP-gamma-S to obtain a first mixture; S2, incubating the first mixture along with total protein which is rich in LRR receptor kinase structures so as to obtain a second mixture; S3, incubating thesecond mixture along with PNBM to obtain a third mixture; S4, removing the PNBM in the third mixture to obtain a fourth mixture; S5, incubating the fourth mixture with pretreated protein G agarose toobtain first supernatant; S6, incubating the first supernatant along with a phosphorothioate resistant monoclonal antibody, which is in coupled linkage with the protein G agarose, so as to obtain first sediment; S7, analyzing the first sediment to determine an LRR receptor kinase substrate.

The present invention relates to apparatus and methods for separating and detecting enzyme substrates using separation gels. For example, the apparatus and methods can be used to separate and detect kinase substrates for further analysis. The apparatus and methods can also be used to detect enzyme inhibitors, such as kinase inhibitors.