59 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 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.

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 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.

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.

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.

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.

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

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.

The invention relates to human recombinant [beta]-interferon fusion protein, which is a fusion protein of human [beta]-interferon and an anti-human serum albumin heavy chain single-domain antibody. The amino acid sequence of the fusion protein is represented as the Seq ID No.1 and the amino acid sequence of the anti-human serum albumin heavy chain single-domain antibody is represented as the Seq ID No.2. In the invention, by means of construction of the fusion expression cloning of the anti-human serum albumin heavy chain single-domain antibody and the [beta]-interferon and introduction into a host cell for expression, a heavy chain single-domain antibody-[beta]-interferon fusion protein having a long acting time is obtained.

The invention relates to sequences of variable regions of an anti-GFAP (glial fibrillary acidic protein) monoclonal antibody. The sequences comprise a nucleotide sequence CM10330-VH and a nucleotide sequence CM10330-VL. A method for preparing the sequences comprises the following steps of: immunizing a mouse by taking a GFAP protein as an antigen, detecting to obtain the mouse of which the corresponding antibody expression is positive, separating the spleen cell of the mouse, fusing the spleen cell with the myeloma cell, culturing the spleen cell and the myeloma cell in an HAT culture medium, detecting to obtain the positive antibody-expression cloning, extracting the total RNA (ribonucleic acid) of the positive hybridoma cell, carrying out inverse transcription by taking the mRNA (messenger ribonucleic acid) in the total RNA as a template to obtain the cDNA (complementary deoxyribonucleic acid) of the gene of the corresponding antibody, obtaining the heavy-chain variable region and the light-chain variable region of the corresponding antibody by using a specific primer through PCR (polymerase chain reaction), and cloning and testing the sequences.