1328 results about "Expression protein" patented technology

The present invention provides methods for identifying targets of a drug in a cell by comparing (i) the effects of the drug on a wild-type cell, (ii) the effects on a wild-type cell of modifications to a putative target of the drug, and (iii) the effects of the drug on a wild-type cell which has had the putative target modified of the drug. In various embodiments, the effects on the cell can be determined by measuring gene expression, protein abundances, protein activities, or a combination of such measurements. In various embodiments, modifications to a putative target in the cell can be made by modifications to the genes encoding the target, modification to abundances of RNAs encoding the target, modifications to abundances of target proteins, or modifications to activities of the target proteins. The present invention also provides methods for drug development based on the methods for identifying drug targets.

Compositions, methods, and kits are provided for efficiently generating and screening a library of highly diverse protein complexes for their ability to bind to other proteins or oligonucleotide sequences. In one aspect of the invention, a library of expression vectors is provided for expressing the library of protein complexes. The library comprises a first nucleotide sequence encoding a first polypeptide subunit; and a second nucleotide sequence encoding a second polypeptide subunit. The first and second nucleotide sequences each independently varies within the library of expression vectors. In addition, the first and second polypeptide subunit are expressed as separate proteins which self-assemble to form a protein complex, such as a double-chain antibody fragment (dcFv or Fab) and a fully assembled antibody, in cells into which the library of expression vectors are introduced. The library of expression vectors can be efficiently generated in yeast cells through homologous recombination; and the encoded proteins complexes with high binding affinity to their target molecule can be selected by high throughput screening in vivo or in vitro.

Disclosed are useful constructs and methods for the expression of proteins using primary translation products that are processed within a recombinant host cell. Constructs comprising a single open reading frame (sORF) are described for protein expression including expression of multiple polypeptides. A primary translation product (a pro-protein or a polyprotein) contains polypeptides such as inteins or hedgehog family auto-processing domains, or variants thereof, inserted in frame between multiple protein subunits of interest. The primary product can also contain cleavage sequences such as other proteolytic cleavage or protease recognition sites, or signal peptides which contain recognition sequences for signal peptidases, separating at least two of the multiple protein subunits. The sequences of the inserted auto-processing polypeptides or cleavage sites can be manipulated to enhance the efficiency of expression of the separate multiple protein subunits. Also disclosed are independent aspects of conducting efficient expression, secretion, and / or multimeric assembly of proteins such as immunoglobulins. Where the polyprotein contains immunoglobulin heavy and light chain segments or fragments capable of antigen recognition, in an embodiment a selectable stoichiometric ratio is at least two copies of a light chain segment per heavy chain segment, with the result that the production of properly folded and assembled functional antibody is made. Modified signal peptides, including such from immunoglobulin light chains, are described.

Methods and compositions are disclosed for inducing differentiation and apoptosis in cells that overexpress Notch proteins. A cell fate determining function of Notch is specifically disrupted at a time when the cell is undergoing differentiation, which causes the cell to undergo apoptosis. The invention includes therapies for tumors that overexpress a Notch protein (such as Notch-1) by inducing differentiation of the cells in the tumor with a differentiation inducing agent, such as HMBA, in combination with an agent that disrupts the function of the Notch protein. At a time during which differentiation has been promoted, and the cell is susceptible to interference with the anti-apoptosis effect of Notch, the function of the Notch protein is disrupted. Disruption of Notch function can be achieved, for example, by a differentiation inducing agent, such as HMBA, combined with antibodies that specifically bind to Notch and inactivate it, for example a monoclonal antibody that recognizes Notch-1 EGF-like repeats 11 and 12, such as monoclonal antibodies A6, C11 or F3. Disruption of Notch function can also be achieved by the expression of antisense oligonucleotides that specifically interfere with expression of the Notch protein on the cell, alone or in combination with antineoplastic agents.

The present disclosure provides combinations of immunotherapeutics and methods for treating medical conditions that are characterized by the lack of an effective immune response, for example as would result following a down-regulation of MHC class I, such as in cancer. The immunotherapeutic compositions of the invention, which can be used to treat the medical conditions, include one or more immunostimulatory antibodies or molecules having specificity for CTLA-4, PD-1, PD-L1, PD-L2, CD40, OX40, CD137, GITR, ILT2, or ILT3, or ligands for these molecules (e.g., an isolated fully-human monoclonal antibody) in association with one or more alloantigens, such as, vector(s) capable of expressing protein(s) or peptide(s) that stimulate T-cell immunity against tissues or cells, formulated in a pharmaceutically acceptable carrier. The proteins or peptides may comprise class I major histocompatibility complex (MHC) antigens, β2-microglobulins, or cytokines. The MHC antigen may be foreign to the subject. The MHC antigen may be HLA-B7.

An improved method is disclosed for the lysis of host cells and extraction of proteins of interest therefrom. The method involves, subsequent to expressing the protein in the host cell by a recombinant DNA procedure, using a reagent solution containing an alkylglycoside or an alkylthioglycoside to lyse the cell to release the protein, and to extract the protein of interest from other host cellular components.

Bacteria which co-express protease inhibitors and protease sensitive therapeutic agents, which are surface displayed, secreted and / or released and result in their localized production and maintenance within a target tissue and inactivation outside of the target tissue, thereby increasing therapeutic activity and reducing the systemic toxicity. The bacteria may be attenuated, non-pathogenic, low pathogenic or a probiotic. Protease sensitivity may be further accomplished by engineering protease degradation sites within the therapeutic agents, further enhancing the inactivation outside of the target tissue while retaining activity within the target tissue through co-expression of a protease inhibitor. Novel chimeric proteins secreted by bacteria, including chimeric toxins targeted to neoplastic cells, tumor matrix cells and cells of the immune system, and combination therapies of these protease inhibitor:chimeric toxin-expressing bacteria together with small-molecule and biologic agents are also described. Non-conjugative bacteria limiting exchange of genetic material, and antibody resistant bacteria are also provided.

The invention discloses a method for knocking out MSTN (myostatin) genes in a targeted manner by utilizing CRISPR-Cas9. According to the method, multiple species are subjected to homology comparison, two pairs of MSTN gene target sequences are selected, two pairs of DNA double strands which have the sequences different from the target sequences, the same expression protein and the same BsmBi sticky ends are synthesized according to the design principle of PAM of gRNA, the double strands are connected with pPDNA330 plasmids, a recombinant vector carrying MSTN homologous genes of multiple species is obtained and is used for detecting the gene knockout efficiency with a fluorescent protein expression method, carrier-transfected receptor cells, having higher knockout efficiency, of multiple species are selected, gene knockout and verification are performed, and simple, efficient and accurate gene knockout on the MSTN genes of the multiple species is completed.

The invention provides a DNA molecule comprising a multicistronic transcription unit coding for i) a selectable marker polypeptide functional in a eukaryotic host cell, and for ii) a polypeptide of interest, the polypeptide of interest having a translation initiation sequence separate from that of the selectable marker polypeptide, characterized in that the coding sequence for the polypeptide of interest is downstream from the coding sequence for the selectable marker in said multicistronic transcription unit, and the nucleic acid sequence coding for the selectable marker polypeptide comprises a mutation that decreases the translation efficiency of the selectable marker in a eukaryotic host cell. The invention also provides methods for obtaining host cells expressing a polypeptide of interest, said host cells comprising the DNA molecules of the invention. The invention further provides the production of polypeptides of interest, comprising culturing host cells comprising the DNA molecules according to the invention.

The present invention provides a method in which cDNAs that encode signal sequences for secreted or membrane-associated proteins are isolated using a fusion protein that directs secretion of a molecule that provides antibiotic resistance, e.g., .beta.-lactamase. The present method allows the isolation of signal peptide-associated proteins that may be difficult to isolate with other techniques. Moreover, the present method is amenable to throughput screening techniques and automation, and especially in validating the presence of the signal sequence via expression of the protein in both prokaryotic and eukaryotic cells. This invention provides a powerful and approach to the large scale isolation of novel secreted proteins.

This invention relates to methods of inhibiting the growth of cancer cells that overexpress a Wnt protein. The methods comprise contacting the cell with an agent that inhibits binding of the Wnt protein to a Frizzled receptor.

The present invention relates to methods and tools for producing large quantities of gamma-carboxylated protein comprising: (i) culturing a cell adapted to express a protein which requires gamma-carboxylation and γ-glutamyl carboxylase in a ratio of at least 10:1, under conditions suitable for expression of both proteins, and (ii) isolating gamma-carboxylated protein.

The present invention relates to a method for display of proteinson spore surface and a method for improving protein with rapidity using the same, which comprises the steps of (i) preparing a vector for spore surface display comprising a gene construct containing a gene encoding spore coat protein and a gene encoding a protein of interest, wherein, when expressed, the gene construct expresses a fusion protein between the spore coat protein and the protein of interest, (ii) transforming a host cell with the vector for spore surface display; (iii) displaying the protein of interest on a surface of a spore of the host cell; and (iv) recovering the spore displaying on its surface the protein of interest.

The invention belongs to the technical field of biological vaccine preparation, and particularly relates to a porcine epidemic diarrhea (PED) recombinant baculovirus gene engineering subunit vaccine, a preparation method and an application thereof. According to the present invention, S1 gene and M gene of the current new PEDV epidemic strain are selected as reference sequences, a baculovirus expression system is adopted to express S1 protein or partial S1 protein and M protein, and the obtained recombinant protein is prepared into a subunit vaccine for effectively controlling PED occurrence; with the PED recombinant baculovirus gene engineering subunit vaccine produced by using the method, the defect of the current PEDV traditional vaccine is solved; and the PED recombinant baculovirus gene engineering subunit vaccine can be used for prevention and treatment of PEDV infections and related diseases caused by PEDV, and can further be used for preparation of coating antigen of PEDV detection antibody ELISA kits.

The present invention relates to expression vectors, wherein each vector comprises at least one gene of interest and a promoter operatively linked thereto wherein each promoter comprises a nucleic acid, whose sequence is randomly mutated with respect to that of the wild-type promoter and cells comprising the same. Methods utilizing either the vectors or cells of this invention, in optimizing regulation of gene expression, protein expression, or optimized gene or protein delivery are described.

The invention discloses a subunit coronavirus vaccine for dimerization-based receptor binding domains and belongs to the technical field of medicine. A baculovirus expresses RBD (receptor binding domain (E367-Y606) of MERS-CoV (middle east respiratory syndrome coronavirus) protein and RBD (R294-F515) of SARS-CoV (severe acute respiratory syndrome coronavirus) in insect cells, the RBDs may form a dimer through cysteine residue at 603 of S (spike) protein or form a dimer through cysteine residue at 512 of the S protein, and purified RBD protein dimer and monomer are used respectively to immunize Bald / c mice. The dimerized RBDs have the advantages that the defect that RBD monomers have poor immunogenicity is overcome and the generation of neutralizing antibodies in against MERS-CoV is increased greatly.

The present invention relates to a method of selecting a protein variant having reduced immunogenicity as compared with the parent protein. This method includes the steps of screening a random peptide display package library with antibodies raised against any protein of interest, sequencing the amino acid sequence of the antibody binding peptides, or the DNA sequence encoding the antibody binding peptides, identifying epitope patterns of a protein by sequence alignment of the reactive peptide sequence, localization of epitope patterns on the primary 3-dimensional structure of the parent protein, defining an epitope area including amino acids situated within 5 Å from the epitope amino acids, and affecting antibody binding to the epitope, changing the localized epitope patterns, or amino acids defining the epitope area of the parent protein by genetic engineering mutations of a DNA sequence encoding the parent protein without impairing functionality of the protein using the emerging epitode database for eliminating amino acid substitutions creating new or duplicating existing epitope patterns, introducing the mutated DNA sequence into a suitable host, culturing the host and expressing the protein variant, and evaluating the immunogenicity of the protein variant using the parent protein as reference. The invention further relates to the protein variant and its use, as well as to a method for producing said protein variant.

The invention provides an anti-CD19 monoclonal antibody, and a preparation method and an application thereof. The anti-CD19 monoclonal antibody can effectively recognize cells with CD19 highly expressed, and can be successfully used for Western Blot detection. An affinity test shows that the monoclonal antibody has the extremely high affinity to target protein.

Therapeutic methods for inhibiting the growth of preneoplastic / neoplastic vertebrate cells that abnormally express MN protein are disclosed. Screening assays are provided for identifying compounds, preferably organic compounds, preferably aromatic and heterocylic sulfonamides, which inhibit the enzymatic activity of MN / CA IX and that are useful for treating patients with preneoplastic / neoplastic disease. Further, the CA IX-specific inhibitors when labeled or linked to an appropriate visualizing means can also be used diagnostically / prognostically for preneoplastic / neoplastic disease, and for imaging use, for example, to detect hypoxic precancerous cells, tumors and / or metastases, by selectively binding to activated CA IX, preferably CA IX activated under hypoxic conditions, and not to inactive CA IX. Such detection of hypoxic conditions can be helpful in determining effective treatment options, and in predicting treatment outcome and the prognosis of disease development. Still further, the CA IX-specific inhibitors can be used therapeutically to selectively target hypoxic cells expressing activated CA IX. The CA IX-specific inhibitors can be labelled or conjugated to radioisotopes for radiotherapy of hypoxic cells. Alternatively, the CA IX-specific inhibitors can be used for gene therapy coupled to vectors for targeted delivery to hypoxic preneoplastic / neoplastic cells expressing activated CA IX on their surfaces. In an alternative mode of the invention, CA IX-specific inhibitors may be used therapeutically to target acidic conditions of a tumor, e.g., to increase pHe in order to enhance the efficacy of weak base chemotherapeutic drugs.

The invention discloses a recombinant human collagen and an application thereof. An amino acid sequence of the protein is shown as SEQ ID NO. 3, the nucleotide sequence of the protein-coding gene is shown as SEQ ID NO. 1. The recombinant human collagen of the invention has very good hydrophilicity and stability, and the amino acid composition thereof is 100% identical to the corresponding amino acid sequence of the natural collagen, and the amount of expressed protein can account for about 25% of the total protein of the thalline, per milliliter of the bacteria liquid precipitate contains 0.25mg of the target protein, and the production cost is very low and the cycle is short. The prepared collagen is applied to the human body without immunological rejection and allergic reaction, and canbe widely applied to the biomedicine and cosmetics industries.

The invention discloses a novel coronavirus N-S dominant epitope fusion protein, a preparation method, application, an expression protein, a microorganism, application and a kit, and belongs to the technical field of genetic engineering. The novel coronavirus N-S dominant epitope fusion protein is a novel coronavirus N protein and S protein dominant epitope fusion protein. The preparation method comprises the following steps: firstly, constructing a fusion expression antigen by an S protein dominant epitope segment antigen and an N protein dominant epitope segment antigen of a novel coronavirus; then removing rare codons through gene optimization to obtain an optimized gene sequence; and finally, obtaining a soluble high-activity antigen with efficient expression. The novel coronavirus N-Sdominant epitope fusion protein disclosed by the invention has soluble expression, high yield, high purity and good activity, and is suitable for accurate diagnosis and detection of patients with thenovel coronavirus.

The present invention relates to the production of proteins in a cell or host cell. The invention uses a TRAnscription Pause (TRAP) sequence to enhance a protein expression characteristic of a protein expression unit. The TRAP sequence is thought to prevent, at least in part, formation of antisense RNA or to, at least in part, prevent transcription to enter the protein expression unit. In one embodiment, the invention provides a method for expression of at least one protein of interest in a cell comprising providing the cell with at least one protein expression unit that comprises a promoter functionally linked to an open reading frame encoding at least one protein of interest, characterized in that the protein expression unit further comprises at least one TRAP sequence and wherein the TRAP sequence is functionally located downstream of the open reading frame and at least in part prevents formation of antisense RNA. In another embodiment, the TRAP sequence is functionally located upstream of the promoter and at least in part prevents transcription to enter the expression unit. Preferably, the expression protein unit further comprises at least one STabilizing Anti-Repressor sequence.

The invention discloses a biological product for preventing novel coronavirus (COVID-19). The biological product can be a gene vaccine or gene medicine, wherein the gene vaccine adopts a human type-5adenovirus with deletion of E1 and E3 genes as a vector for carrying an S1 protein antigen for expressing a Spike S1 subunit of the novel coronaviruses or simultaneously carrying an S1 expression protein antigen and N protein antigen. Through in-vivo expression of antigen proteins, the immune reaction of a body upon the novel coronavirus can be stimulated, and the effect of preventing infection and propagation of the novel coronavirus.

The invention discloses a method of determining protease activity, in real time, using fluorescence polarization technology. In particular, the invention provides vectors and a method for their use, which expresses uncharacterized proteins conjugated to a fluorescence tag, which binds specifically to a fluorescent ligand. Cleavage of the recombinant protein results in a fragment of the expressed peptide and results in a change in fluorescence polarization of the fluorophore. The rate of change in fluorescence polarization can be measured in real time and is equivalent to the rate of protease cleavage.