35 results about "Staphylokinase" patented technology

The invention relates to a polyethylene glycol (PEG) modification method. The method is capable of realizing selective modification of carbon terminal carboxyl groups of proteins and polypeptides, and each protein molecule and polypeptide molecule is connected with one PEG molecule. According to the method, staphylokinase is used as a model protein. The polyethylene glycol (PEG) modification method comprises following main steps: (1) under slightly acidic conditions (pH 5.0), carboxyl groups of staphylokinase and amino groups of cystamine are connected; and (2) reduction of disulfide bonds of cystamine is realized using a reducing agent, and generated sulfhydryl groups are reacted with methoxy polyethylene glycol-maleinimide (mPEG-Mal). pKa value of the carbon terminal carboxyl groups of staphylokinase is 2.1 to 2.4, pKa value of side chain carboxyl groups of aspartic acid is 3.7 to 4.0, and pKa value of side chain carboxyl groups of glutamic acid is 4.2 to 4.5, so that under the conditions with a pH value of 5.0, PEG is capable of realizing selective modification of the carbon terminal carboxyl groups of staphylokinase. The method is mainly used for PEG selective modification of the carbon terminal carboxyl groups of the proteins and the polypeptides; a novel PEG modification method is provided; and an advantage of the method is that uniformity of modification sites of the PEG modification product is excellent.

New thrombolytic protein molecules such as recombinant staphylokinase or streptokinase, urokinase, tissue plasminogen activator and the like, and suitable variants thereof, for targeting to brain tissue or any other tissue by either fusing to, or by synthesizing the candidate thrombolytic molecule(s) with a protein sequence comprising a strong amphipathic alpha helix containing protein transduction domain. Thrombolytic protein molecule(s) so engineered with the protein transduction domain is useful for enhanced uptake of such protein thrombolytic molecule(s) across the cell membranes and tissues including the blood brain barrier and find their use in the treatment of vascular thrombosis including cerebrovascular disorders caused by cerebral thrombosis or cerebral haemorrhage when used a as a therapeutic. The design and processes for cloning, expression, purification and protein transduction of such proteins across cell membranes.

The invention discloses a protein structure of a low-immunogenicity recombinant staphylokinase mutant and a preparation method thereof. The invention relates to the molecular structure of the low-immunogenicity recombinant staphylokinase mutant. The preparation method comprises: directly obtaining the expression plasmid of the recombinant staphylokinase mutant by a polymerase chain reaction (PCR)site-directed mutagenesis technique; transforming Escherichia coli; performing fermentation culture; inducing expression; breaking thalli; centrifuging; collecting supernate; obtaining pure protein by continuous three-step chromatographic purification; and subjecting the pure protein to freeze drying to obtain the product. The obtained staphylokinase mutant has much lower immunogenicity, retains fibrinolysis activity for dissolving thrombi and can be used in preparation of medicaments for treating thromboembolic diseases.

The invention discloses a method for expressing and preparing staphylokinase (Staphylokinase, SAK), comprising: 1. Construction of staphylokinase fusion protein expression engineering bacteria: constructing a recombinant plasmid containing the gene sequence of SEQ NO.1, and introducing the recombinant plasmid into Escherichia coli to obtain fermentation strains. 2. The preparation method includes: a. fermenting and expressing a soluble fusion protein having an amino acid sequence such as SEQ NO.2 by engineering bacteria; b. absorbing the fusion protein in step a with a nickel ion affinity gel column to extract the fusion protein; c. Dilute the fusion protein in step b with enterokinase, dilute appropriately, pass through the gel column in step b, and collect the permeate; d, concentrate the permeate through ultrafiltration in step c, and pass through the Q gel column to obtain the staphylokinase stock solution . The method of the invention has simple and reliable steps, and is especially suitable for large-scale production. The staphylokinase obtained by the method of the invention has high purity, high specific activity and good thrombolytic activity; it provides a new way for producing medicinal staphylokinase.

The invention discloses a staphylokinase mutant, wherein cysteine is introduced to the C-end of the staphylokinase mutant. Compared with the wild type staphylokinase, the C-end of the mutant has three more amino acids, namely glycine-glycine-cysteine (Gly-Gly-Cys). The invention also discloses a preparation method of the dimer of the staphylokinase mutant. The dimerization method of the staphylokinase is that the two maleimide groups of the homotype bifunctional binder 1,4-dimaleimide butane and the cysteine at the C-end of the staphylokinase are utilized to form a stable covalent bond. The invention also discloses a preparation method for realizing the N-end site-specific modificaton of polyethylene glycol by using the dimer of the staphylokinase mutant. Under special conditions, methoxypolyethylene glycol-propionaldehyde is connected with one N-end of the staphylokinase dimer molecule through the covalent bond. The biological activity of the polyethylene glycol modified product of the staphylokinase dimer is higher than that of the modified product of the staphylokinase monomer.

The present invention relates to a nucleotide sequence of expression cassette OXY-1 of SEQ ID No. 1, a modified staphylokinase SAK-2 gene of SEQ ID No. 2, a peptide sequence of modified staphylokinase SAK-2 gene, of SEQ ID No. 3, three plasmids having International Deposition Nos. BPL-0019, BPL-0020, and BPL-0021, and their corresponding three recombinant E. Coli; also invention relates to a process for over-producing staphylokinase and its analogues by modulating level of oxygen of its growth medium in a host system, and lastly, a method of dissolving blood clot in a subject in need thereof.

The invention relates to an anti-bloodpatelet aggregation, low-immunogenicity recombinant staphylokinase mutant and its preparing method. The invention analyses the structures of recombinant staphylokinase monomer and micro-lumbrokinase-staphylokinase-micro- lumbrokinase compound crystal, and designs a novel molecular structure of staphylokinase mutant, which is recombined with a prokaryotic expression carrier pLY-4 after constructing mutant gene by using PCR fixed-point mutation, converting Escherichia coli, and screening high-expression engineering bacteria; by fermenting and amplifying, crushing the bacteria, centrifuging and collecting, then making two-step purification, and freeze-drying and obtaining the product, which basically maintains fibrinolytic activity of wild staphylokinase, can inhibit the bloodpatelet aggregation and has remarkable function of preventing and curing blood acid and its immunogenicity is remarkably reduced.

The invention belongs to the technical field of genetic engineering and particularly relates to pegylated staphylokinase and preparation and application thereof. The pegylated staphylokinase structurally comprises polyethylene glycol and mutated staphylokinase which are coupled with each other. Compared with current similar products, the pegylated staphylokinase has the advantages that the immunogenicity of natural staphylokinase is effectively reduced, and sufficient thrombolytic activity is maintained.

The invention discloses a protein structure of a low-immunogenicity recombinant staphylokinase mutant and a preparation method thereof. The invention relates to the molecular structure of the low-immunogenicity recombinant staphylokinase mutant. The preparation method comprises: directly obtaining the expression plasmid of the recombinant staphylokinase mutant by a polymerase chain reaction (PCR)site-directed mutagenesis technique; transforming Escherichia coli; performing fermentation culture; inducing expression; breaking thalli; centrifuging; collecting supernate; obtaining pure protein by continuous three-step chromatographic purification; and subjecting the pure protein to freeze drying to obtain the product. The obtained staphylokinase mutant has much lower immunogenicity, retains fibrinolysis activity for dissolving thrombi and can be used in preparation of medicaments for treating thromboembolic diseases.

This invention belongs to the biology project pharmacy filed, which provides a Staphylokinase (SAK) with low pyrogen and it's preparing method. The Staphylokinase has low pyrogen, high purify and strong activity, which is completely fit for the medicinal demand. This invention includes the following steps: a breaking up the material containing with the Staphylokinase and distilling the floating liquid; b. sieving the product from step a with the DEAD gelatin rob, then removing salt and condensing; c. sieving the product form step b with CM gelatin rob, collecting the sample apex, condensing the washing liquid and transforming the medium; d. sieving the product from step c with the Q gelatin rob to obtain the Staphylokinase. The Staphylokinase of this invention has good bolt dissolving effect, and it is safe and efficient for users..

The invention provides a method for preparing recombinant human octoplasmin, and belongs to the technical field of biological medicine. The method comprises the following steps: (1) preparing staphylokinase subjected to enzyme digestion: taking recombinant staphylokinase for enzyme digestion, and separating and purifying an enzyme digestion substance to obtain staphylokinase subjected to enzyme digestion; wherein the amino acid sequence of the staphylokinase subjected to enzyme digestion is as shown in SEQ ID NO: 3; and (2) preparing the recombinant plasmin: adding staphylokinase subjected to enzyme digestion into the recombinant plasminogen for enzyme digestion, and separating and purifying an enzyme digestion substance to obtain the recombinant plasmin. It is found for the first time that the impurity A in the recombinant plasmin can be effectively removed through the method, the purity of the recombinant plasmin is improved, and potential stability and safety risks of drugs in clinical application are eliminated. The method disclosed by the invention is low in cost, simple to operate and suitable for industrial production.

The invention provides an RGD-recombinant staphylokinase-human alpha microglobulin fusion protein and its preparation and application. The amino acid sequence of the RGD-recombinant staphylokinase-human α-microglobulin fusion protein of the present invention is shown in SEQ ID NO.12. The fusion protein of the present invention is obtained by the following preparation method: the target gene fusion fragment is obtained by overlapping extension PCR method; the target gene fusion fragment is inserted into the expression vector to construct a recombinant expression plasmid; the constructed recombinant expression plasmid is transformed into Escherichia coli, and in Escherichia coli High-efficiency expression; separation and purification of fusion protein. Compared with existing similar products, the fusion protein of the invention not only has high-efficiency thrombolytic and anticoagulant activity, but also has low immunogenicity. At the same time, the fusion protein of the present invention also overcomes some existing shortcomings of reduced activity and insoluble expression of RGD-recombinant staphylokinase.

The invention discloses a staphylokinase and an expression vector thereof. The staphylokinase is polypeptide with amino acid sequence shown in SEQ ID No: 1, and the gene coding sequence thereof is shown in SEQ ID No: 2. The invention also discloses an expression vector containing a staphylokinase gene. The thrombolysis of the staphylokinase has strong effect, so that the staphylokinase can be used for preparing medicine for remedying cardio-cerebrovascular diseases, in particular to medicine for remedying myocardial infarction and other thrombotic diseases; the thrombolysis activity of the staphylokinase is high, and the activity rate thereof is 4.0*10<4>AU / mg; in addition, the expression amount of the expression vector of the aphylokinase is high, and the expression amount of the aphylokinase accounts for more than 45 percent of total protein of thallus.

A freeze-dried preparation of recombinant glucokinase in the form free dried powder, injection, lipoplasm, or microcapsule for thrombolytic purpose contains the glucokinase with mutation of amino acids at positions 7, 3 and 43, and one or more of mannitol, phosphate, EDTA and sodium chloride. Its preparing process is also disclosed.