62 results about "Expression cloning" patented technology

The invention provides a genetic screening method for identifying a transfected cell expressing the polypeptide of interest. The methods allows for high throughput screening of recombinant cells for elevated levels of expression of the polypeptide of interest. The invention also provides capture media, formulations and methods of making and using thereof.

The invention discloses an expression cloning method and device capable of realizing real-time interaction with a virtual character and belongs to the fields such as computer graphics and virtual reality. The method includes the following steps that: 1, modeling and skeleton binding are performed on the virtual character; 2, the basic expression base of the virtual character is established; 3, expression input training is carried out: the maximum displacement of facial feature points under each basic expression is recorded; 4, expression tracking is carried out: the facial expression change of a real person is recorded through motion capture equipment, and the weights of the basic expressions are obtained through calculation; 5, expression mapping is carried out: the obtained weights of the basic expressions are transferred to the virtual character in real time, and rotation interpolation is performed on corresponding skeletons; and the real-time rendering output of the expression of the virtual character is carried out. With the method adopted, the expression of the virtual character can be synthesized rapidly, stably and vividly, so that the virtual character can perform expression interaction with the real person stably in real time.

The present invention is directed to a method for detection, characterization and isolation of nucleic acids encoding proteins of a desired property, such as a particular cellular localization. The invention further provides for rapid expression of such proteins or glycoproteins in mammalian cells and for facilitated purification of the novel secreted proteins or glycoproteins. Further, the invention provides for radioactive labelling of the novel proteins or glycoproteins, for rapid identification of sites of binding including animals and for rapid production of infective viral vectors for use in gene transfer.

The present invention is directed to a method for detection, characterization and isolation of nucleic acids encoding proteins of a desired property, such as a particular cellular localization. The invention further provides for rapid expression of such proteins or glycoproteins in mammalian cells and for facilitated purification of the novel secreted proteins or glycoproteins. Further, the invention provides for radioactive labelling of the novel proteins or glycoproteins, for rapid identification of sites of binding including animals and for rapid production of infective viral vectors for use in gene transfer.

Methods are provided for isolation of DNA sequences encoding proteins with properties of interest by means of expression cloning in filamentous fungal host cells. The isolated DNA sequences are useful in processes for producing the proteins of interest.

The present invention relates to methods for producing a recombinant polypeptide of interest, the method comprising the steps of: a) providing a polynucleotide library encoding one or more polypeptides of interest, wherein the library was prepared in an expression cloning vector comprising at least the following elements: i) a polynucleotide encoding a selectable marker; ii) a fungal replication initiation sequence, preferably an autonomously replicating sequence (ARS); and iii) a polynucleotide comprising in sequential order: a promoter derived from a fungal cell, a cloning-site into which the library is cloned, and a transcription terminator; b) transforming a mutant of a parent filamentous fungal host cell with the library, wherein the frequency of non-homologous recombination in the mutant has been decreased compared to the parent; c) culturing the transformed host cell obtained in (b) under conditions suitable for expression of the polynucleotide library; and d) selecting a transformed host cell which produces the polypeptide of interest.

A method for the recovery of full-length cDNAs for plant genes containing introns is described. The method utilizes Agrobacterium-mediated transient expression coupled with an RT-PCR assay to facilitate expression cloning of processed transcripts. Subsequent expression of intron-less cDNAs in a suitable prokaryotic or eucaryotic host provides for direction functional testing of the encoded gene product.

The present invention is a substantially purified sortase-transamidase enzyme from Gram-positive bacteria, such as Staphylococcus aureus. A specific sortase-transamidase enzyme disclosed has a molecular weight of about 29,076 daltons and catalyzes a reaction that covalently cross-links the carboxyl terminus of a protein having a sorting signal to the peptidoglycan of a Gram-positive bacterium, where the sorting signal has a a motif of NPQ / KTN / G therein. Variants of the enzyme, methods for cloning the gene encoding the enzyme and expressing the cloned gene, and methods of use of the enzyme, including for screening for antibiotics and for display of proteins or peptides on the surfaces of Gram-positive bacteria, are also disclosed.

The present invention relates to the field of molecular cloning and to the field of expression cloning in higher, eukaryotic cells. In particular, the present invention relates to a method for fast and reliable identification of vectors and vector DNA which will be capable of providing a desired expression product if a eukaryotic host cell is transfected with the vector DNA.

The present invention provides a process and methods for producing asymmetric antibodies in a mammalian expression system. The asymmetric antibodies are transiently or stably expressed and in cells that stably express the asymmetric antibody, following a rapid 2-step process of stable pool to clone, a highly pure asymmetric antibody expressing clone can be identified at a success frequency that permits for screening of tens of clones rather than thousands. The asymmetric antibodies are produced at a high titre and with a high level of purity with no contaminating homodimer antibodies following protein A purification with a step yield of near 100%. Typical downstream purification processes employ standard hydrophobic interaction chromatography (HIC) and / or cation exchange (CEX) resins and the antibody is stable within a wide dynamic range of buffer pH (4-8) and within the requirements for manufacturing antibodies for pre-clinical and clinical applications.

The invention relates to a glutamine synthetase expression vector which can be amplified and have two expression cassettes. The main components of the glutamine synthetase expression vector include the following six parts: a first expression cassette component, a second expression cassette component, an f1 replicon, a glutamine synthetase expression cassette component, an ampicillin beta lactamase hydrolysis expression cassette component and a ColE1 replicon, wherein a strong enhancer / promotor CMV (Cytomegalovirus) and an immediate early enhancer / promotor are adopted to the first expression cassette component and the second expression cassette component; a weak promotor / enhancer SV40 is adopted to the glutamine synthetase expression cassette component, so that the expression of glutamine synthetase is reduced, and the screening of high-expression cloning is facilitated; and expression protein coding genes are respectively cloned to the expression vector through a multiple cloning site A and a multiple cloning site B. The glutamine synthetase expression vector disclosed by the invention is suitable for simultaneously expressing 1-2 proteins efficiently in mammalian cells and especially suitable for expressing antibody proteins.

The invention relates to an expression vector containing a green fluorescent protein gene. The green fluorescent protein gene is a gene sfGFP, wherein the nucleotide sequence of the gene sfGFP is shown in SEQ ID NO: 1. The invention further relates to a construction method of the expression vector containing the green fluorescent protein gene and application of the expression vector containing the green fluorescent protein gene in positive clone screening. According to the expression vector containing the green fluorescent protein gene and the construction method and application thereof, the characteristic that the sfGFP can complete super-folding and illuminate under various conditions is utilized, the advantages of luminous power and luminous intensity of the sfGFP are obviously stronger than those of ordinary proteins, such as GFP (Green Fluorescent Protein), EGFP (Enhanced Green Fluorescent Protein) and the like, and are 1.6 times the EGFP, and obvious green fluorescent light can be observed by unaided eyes, so that the screening of positive clones during protein expressed cloning can be facilitated, and the fast and accurate screening for the positive clones is facilitated.

The invention discloses an application and method for squalene synthase gene overexpression of medicago sativa. The method includes steps: (1) primer design; (2) vector pEASY-T3 plasmid cloning, wherein pGEX-4T-1V is used for prokaryotic expression, and escherichia coli E.coli Trans5alpha and E.coli Transetta are used for gene cloning and expression; (3) cloning of full-length cDNA of medicago sativa squalene synthase gene MsSQS; (4) biological information analysis of an MsSQS sequence; (5) expression analysis of the squalene synthase gene MsSQS; (6) determination of squalene synthase content;(7) methyl jasmonate induced saponin content determination; (8) subcellular localization analysis; (9) prokaryotic expression; (10) MsSQS gene over-expression vector construction and medicago sativagenetic transformation. By establishment of an efficient genetic transformation system, overexpression of the squalene synthase gene in medicago sativa is realized, the content of total saponins in transgenic medicago sativa is increased, and breeding materials are provided for cultivating high-saponin-content medicago sativa varieties.

A method is described for regulating gene expression related to iron metabolism to ameliorate diseases that include sickle cell disease, cancers, neurodegenerative diseases, Friedreich's ataxia and other neuromuscular disorders, and atherosclerosis. This approach is illustrated by recent findings that show that ferritin-H, an iron-binding protein that is present in cell nuclei, can repress the human beta-globin gene, the gene that is mutated in sickle cell disease. Increased expression of ferritin-H or a related ferritin-family peptide, given to effected cells either as the peptide itself (or a part thereof), as an expression clone of the ferritin-H-subfamily gene, or via a gene regulator that increases expression of the ferritin-H-subfamily gene itself, prevents or ameliorates expression of the disease state in disorders where increased availability of iron is implicated in the etiology of the disease, including those named above.

An expression carrier for effectively screening target protein is prepared through introducing an internal ribosome to the eukaryotic expression carrier containing CMV promoter and dhfr amplification system, linking the fusion gene of anti-erbB2-seFv-Fc gene and IL-2 to the neomycin-resistant gene, site mutation to Neo gene by PCR, and configuring the expression carrier pCMV-HFI-mNeo.

The invention provides cloning of a tobacco root-specific promoter and application thereof to a transgenic plant. The tobacco root-specific promoter at least comprises a nucleotide sequence showed in SEQIDNo.1. The method for cloning the tobacco root-specific promoter comprises the following steps: building an SSH library; manufacturing a probe; screening a specific gene by hybridization in the library; identifying spatial expression of the specific gene by RT-PCR; and cloning a full-length gene sequence and the upstream specific promoter. In the invention, the tobacco root-specific promoter is applied to the transgenic plant. The NtR12 root-specific promoter of the invention can improve the safety of tobacco transgenosis and effectively solve rhizome pests and diseases during the process of planting tobacco.

The invention provides a genetic screening method for identifying a transfected cell expressing the polypeptide of interest. The methods allows for high throughput screening of recombinant cells for elevated levels of expression of the polypeptide of interest using methylcellulose comprising fluorescent protein A or G to improve detection and cloning. The invention also provides capture media, formulations and methods of making and using thereof.

Described herein are methods of expression cloning of components of signaling pathways that activate a transcription factor of interest. The methods are efficient for identifying modulators of transcription factors. The modulators can then be screened further or used directly to develop therapeutics.

The invention provides a eukaryotic expression vector capable of selecting high expression clones conveniently and high efficiently. The eukaryotic expression vector is obtained via structure modification by taking pBudCE4.1 carrier as a basic carrier; peptide chain elongation factor gene promoter (P) on the basic carrier, and multiple cloning sites on the downstream of the peptide chain elongation factor gene promoter (P) are replaced by an expression element composed of attenuated SV40 promoter and dihydrofolate reductase gene on the downstream of the attenuated SV40 promoter; multiple cloning sites on the downstream of cytomegalovirus early promoter (P) of the basic carrier are replaced by new multiple cloning sites.

Novel methods for screening and cloning DNA sequences coding for biocidal molecules, and for identifying biocidal molecules involving use of viability staining assay are provided. The methods consist of construction of libraries of DNA from natural and synthetic sources, and expression of cloned DNA in surrogate hosts, identification of DNA clones biocidal to the host cells by viability assay and isolation of the DNA sequences coding for biocidal molecules. Further provided are methods of identifying the biocidal molecules and their use.

A method is described for regulating gene expression related to iron metabolism to ameliorate diseases that include sickle cell disease, cancers, neurodegenerative diseases, Friedreich's ataxia and other neuromuscular disorders, and atherosclerosis. This approach is illustrated by recent findings that show that ferritin-H, an iron-binding protein that is present in cell nuclei, can repress the human .beta.-globin gene, the gene that is mutated in sickle cell disease. Increased expression of ferritin-H or a related ferritin-family peptide, given to effected cells either as the peptide itself (or a part thereof), as an expression clone of the ferritin-H-subfamily gene, or via a gene regulator that increases expression of the ferritin-H-subfamily gene itself, prevents or ameliorates expression of the disease state in disorders where increased availability of iron is implicated in the etiology of the disease, including those named above.

The invention discloses a recombinant staphylococcus aureus ClfA protein vaccine and a construction method of a eukaryotic expression engineering cell line of the protein vaccine, and belongs to the field of biological pharmacy. The gene sequence of staphylococcus aureus ClfA is SEQ ID NO. 1. The construction method mainly includes the steps: separating and identifying the staphylococcus aureus; extracting genome DNA (deoxyribonucleic acid) of the staphylococcus aureus; designing a primer amplification ClfA gene sequence by taking the extracted staphylococcus aureus genome DNA as a template; connecting the ClfA sequence with pEASYR-T1 Simple Cloning Vector and performing amplification on DH5 alpha; extracting plasmids and respectively performing double enzyme digestion and connection for the ClfA and pcDNA 3.1 V5-His B to obtain recombinant plasmids ClfA-pcDNA 3.1 V5-His B; converting connection recovery products into Escherichia coli DH5 alpha; screening positive clones and extracting the recombinant plasmids ClfA-pcDNA 3.1; transfecting human breast cancer cells MCF-7 by the aid of lipidosome 2000 (lippo2000); screening positive cell clones and high expressing clones by G-418. The construction method can solve the problem that protein products easily form inclusion bodies and are not easily purified when procaryotic organism expression long-sequence genes encode high-molecular-weight protein products.

The invention relates to a promoter DNA sequence highly suited in an improved expression cloning method for isolation of DNA sequences comprising a DNA sequence encoding a protein of interest in a host cell and to the improved expression cloning method wherein use is made of this promoter. The isolated DNA sequences are useful in processes for producing a protein of interest.

The invention provides a phosphate solubilizing gene psg I-13. The nucleotide sequence of the phosphate solubilizing gene psg I-13 is as shown in SEQ ID No.1. The nucleotide sequence of a protein coded by the phosphate solubilizing gene psg I-13 is as shown in SEQ ID No.2. The phosphate solubilizing gene psg I-13 cloned through heterologous expression can be used for efficiently generating organic acids, has a function of converting indissoluble phosphate into effective phosphate and provides a base material for increasing the utilization rate of a phosphate fertilizer by using a molecular biology means.

Methods of expression cloning where a cDNA construct expresses a tagged polypeptide for a biochemical activity of interest are described.

The invention discloses a complete extracellular fragment of a disintegrin-metalloproteinase ADAM15 obtained through recombinant expression of a eukaryotic cell, a preparation method and applications thereof, and aims to provide an ADAM15 protein capable of being applied to production and research. The recombinant ADAM15h protein provided by the invention is an ADAM15h fusion protein which is obtained by converting and expressing a host cell line by pSCT-ADAM15h. The invention provides a preparation method of the recombinant ADAM15h fusion protein. The preparation method comprises the following steps: converting a host cell line by using pSTC-ADAM15h, culturing the converted host cell line, and screening the recombinant ADAM15h high expression clone so as to obtain the recombinant ADAM15h fusion protein. The invention also provides a method for purifying ADAM15h fusion protein in cell culture supernatant and a method for detecting the enzyme activity of recombinant ADAM15h by cutting a fluorescent substrate.

The invention discloses an expression cloning method and device capable of realizing real-time interaction with a virtual character and belongs to the fields such as computer graphics and virtual reality. The method includes the following steps that: 1, modeling and skeleton binding are performed on the virtual character; 2, the basic expression base of the virtual character is established; 3, expression input training is carried out: the maximum displacement of facial feature points under each basic expression is recorded; 4, expression tracking is carried out: the facial expression change of a real person is recorded through motion capture equipment, and the weights of the basic expressions are obtained through calculation; 5, expression mapping is carried out: the obtained weights of the basic expressions are transferred to the virtual character in real time, and rotation interpolation is performed on corresponding skeletons; and the real-time rendering output of the expression of the virtual character is carried out. With the method adopted, the expression of the virtual character can be synthesized rapidly, stably and vividly, so that the virtual character can perform expression interaction with the real person stably in real time.

The invention provides a culture medium for cloning formation of Chinese hamster ovary cells. The culture medium provided by the invention does not contain serum or animal-derived components. The cloning culture medium provided by the invention can enable the CHO cells to be more compact in cloning agglomeration and less in diffusion, the growth speed of cloning is fast, and expression clones suitable for production and use purposes can be more efficiently selected.

The invention provides a novel expression cloning technique referred to as a matrix analysis of gene expression in cells (MAGEC) that allows for the indexed introduction, and analysis of nucleic acids in a host cell. While normally one takes cells attached to a surface followed by contacting the cells with heterologous DNA under conditions favoring the uptake of the heterologous DNA, the present invention, in sharp contrasts, proposes affixing (depositing) a nucleic acid-containing mixture onto a suitable surface and thereafter contacting suitable host cells (target cells) with the DNA-containing markings under conditions favoring uptake by the cells of the heterologous expression vector comprising a the target nucleic slide acid molecule. The method enables one to further characterize the gene product(s) of a known gene and unknown in a high-throughput assay format. It essentially allows for the identification of a gene based upon the function of its gene product.