119 results about "Proinsulin" patented technology

The present invention discloses a treatment for syndrome-X, and resulting complications, that include hyperlipidemia, hypertension, central obesity, hyperinsulinemia and impaired glucose intolerance. Diabetic complications include excess proinsulin levels.

Methods are provided for reducing serum proinsulin levels, lessening post-prandial pancreatic stress, and reducing risk factors for atherosclerosis in subjects with diabetes mellitus, type 2. The method includes administration of insulin in a manner that mimics the meal-related first phase insulin response, using a dose sufficient to reduce serum levels of proinsulin. In some embodiments of the method insulin administration is commenced early in the course of the disease. Mimicking first phase kinetics, peak serum insulin levels can be reached within about 18 minutes of administration. In increasingly preferred embodiments peak serum insulin levels can be reached within about 15, 12, or 10 minutes of administration. Serum insulin levels return to baseline within about two hours of administration.

The present invention relates to a specific C-peptide assay method which can eliminate all interference due to proinsulin and its intermediates, in particular des-31,32-proinsulin and/or des-64,65-proinsulin. It also relates to antibodies for carrying out this assay.

The invention provides a molecule (Preproinsulin) of human proinsulin with a novel N-terminal expressed peptide sequence or analogs of the human proinsulin, a method for producing human insulin by using the molecule, and processes for building related expression vectors and engineering cells and expressing and purifying human proinsulin. The DNA sequence of the human proinsulin coded by the N-terminal expressed peptide sequence or the analogs of the human proinsulin is first introduced into a prokaryotic expression vector and then transferred into an escherichia coli to express the molecule in form of an inclusion body. The invention has the advantages that: the product has high expression amount and is easy to purify; the preparation method avoids the use of CNBr; and the process for processing the recombinant insulin is simple.

Disclosed is a method for obtaining glucose-regulated expression of active insulin in the cells of a mammalian subject. The method involves delivering into the subject a genetic construct comprising a coding sequence for a human proinsulin operably connected a promoter functional in the host cells. The construct includes a glucose responsive regulatory module having at least one glucose inducible regulatory element comprising a pair of CACGTG motifs linked by a five base nucleotide sequence, which confers glucose inducible expression of the proinsulin coding sequence. To ensure proper processing of the proinsulin to active insulin, the coding sequence was modified to direct the synthesis of a mutant proinsulin polypeptide having amino acid sequences that can be cleaved to mature insulin in suitable host cells, such as hepatocytes.

The invention discloses a preparation method of recombinant human insulin, which relates to the protein polypeptide type medicine field. The technical problem needed to be solved by the invention aims at providing a preparation method for Escherichia coli to express the recombinant human insulin in order to overcome the technical disadvantages in the prior art of using the poisonous and harmful substance CNBr and having a complex production process. The preparation method for the recombinant human insulin of the invention comprises the steps of: thallus crashing, inclusion body dissolving, proinsulin sulfonating, purifying proinsulin, and proinsulin renaturation, enzyme cutting transmission, etc., and the preparation method is characterized in that during the inclusion body dissolving process, the -SH of proinsulin is transformed into -SSO3<->, and then human insulin is obtained through purifying the sulfonated proinsulin, reduction renaturation and enzyme cutting transmission by positive ion, and then through the negative ion exchange layer purification. The preparation method of the recombinant human insulin of the invention does not use the CNBr and has simple technique with high human insulin yield coefficient and purity.

The invention discloses a preparation method for insulin aspart through recombinant expression by using yeast, and concretely relates to a preparation method for insulin aspart through recombinant expression by using pichia yeast. Concretely, the method comprises the following technological process: effectively secreting and expressing human aspart proinsulin, performing lysyl endopeptidase single enzyme digestion to obtain insulin aspart deleting B30, coupling with a threoninate, and performing deprotection, anti-phase purification and crystallization. The method is relatively suitable for industrialized preparation of recombinant insulin aspart.

Glargine proinsulin sconstructs that have a modified C-peptide amino acid and/or nucleic acid sequence for producing glargine insulin analogs are provided. Highly efficient processes for preparing the glargine insulin analogs and improved preparations containing the glargine insulin analogs prepared according to the methods described herein are also provided.

The invention provides a human proinsulin chemiluminescence immunoassay kit and a preparation method thereof. The kit provided by the invention comprises an anti-proinsulin reagent 1, an anti-proinsulin reagent 2, a magnetic separation reagent, a proinsulin calibrator, a chemiluminescence substrate, and a washing liquid. The invention also provides a preparation method of the kit. The preparation method comprises the following steps: coupling antibody and biotin, preparing the anti-proinsulin reagent 1, coupling enzyme and antibody, preparing the anti-proinsulin reagent 2, preparing the magnetic separation reagent, preparing the proinsulin correction sample, and preparing the chemiluminescence substrate. Compared to the proinsulin kit existing in the present market, the kit provided by the invention has the characteristics of high detection sensitivity, good repeatability, and low cost.

Provided herein are compositions and methods for the treatment of type 1 diabetes (T1D) in mammalian subjects. The compositions include lactic acid fermenting bacteria (LAB) expressing an IL-2 gene and a T1D-specific self-antigen (e.g., proinsulin (PINS)) gene. Exemplary methods include: orally administering to a mammalian subject, a therapeutically effective amount of the composition. The composition can be administered to the subject mucosally, resulting in delivery of the LAB into the gastrointestinal tract, where the LAB is released. Bioactive polypeptides expressed by the LAB are thus administered via mucosal delivery. The LAB may be selected to deliver a low-dose of IL-2 to the subject. The methods may not require concomitant systemic anti-CD3 antibody treatment. The methods may be suited for subjects possessing residual beta-cell function, e.g., those with recent-onset T1D.

The invention discloses a method for performing high performance size exclusion chromatography (HPSEC) and purifying recombinant human proinsulin renaturation. The method comprises the following steps: (1) performing ultrasonic cell disruption on the proinsulin-containing thallus cell, washing and dissolving the buffer solution in strong denaturant (such as 8mol/L or 6-7 mol/L guanidine hydrochloride) thereby obtaining the recombinant human proinsulin subjected to crude separation; (2) directly feeding the proinsulin-containing denaturated leaching liquor under the optimized chromatographic condition, wherein the purity of target fraction rhPI obtained through one-step chromatography by employing urea concentration linear gradient elute can be over 96 percent, and the mass recovery rate is 56.8 percent; and (3) further desalting the proinsulin-containing original fraction, thereby obtaining the renaturated and purified high-purity and activity fraction. The recombinant human proinsulin obtained by the method can be converted into insulin through digestion on a chromatographic column or digestion in a solution, and the chromatographic process is easy to operate, high in repeatability and easy for large-scale production.

The present invention provides an insulin-specific assay and an assay reagent capable of sensitively and specifically assaying insulin using an antibody having a property of reacting with insulin bound to an anti-insulin antibody while not reacting with insulin not bound to an anti-insulin antibody, without being affected by proinsulin and insulin analogs.

The invention provides a recombinant human proinsulin transpeptidation method and an application in recombinant human proinsulin downstream purification thereof. The recombinant human proinsulin transpeptidation method is characterized by carrying out transpeptidation reaction on purified recombinant human proinsulin to obtain recombinant human insulin, wherein the reaction system of the transpeptidation reaction comprises 10-30g/L of recombinant human proinsulin, 100-200ml/L of dimethyl sulfoxide (DMSO), 63-103g/L of o-tert-butyl threonine tert-butyl ester, 600-800ml/L of 1,4-butanediol, 2-6g/L of trypsin, 0.2-100mmol/L of Ca<2+>, 0.2-100mmol/L of Mg<2+>, and water, and the pH value is 6.5-7.0. The reaction conditions of the transpeptidation reaction comprise a temperature of 27-47 DEG C, and a stirring time of 2-10 h. According to the invention, the recombinant human proinsulin transpeptidation method disclosed herein can be applied in downstream purification of recombinant human proinsulin. The recombinant human proinsulin transpeptidation method disclosed herein shortens the time of the transpeptidation reaction, raises the production efficiency, and can reduce by-products.

Methods for synthesizing proinsulin polypeptides are described that include contacting a proinsulin polypeptide including an insulin polypeptide coupled to one or more peptides by peptide bond(s) capable of being cleaved to yield the insulin polypeptide with an oligomer under conditions sufficient to couple the oligomer to the insulin polypeptide portion of the proinsulin polypeptide and provide a proinsulin polypeptide-oligomer conjugate, and cleaving the one or more peptides from the proinsulin polypeptide-oligomer conjugate to provide the insulin polypeptide-oligomer conjugate. Methods of synthesizing proinsulin polypeptide-oligomer conjugates are also provided as are proinsulin polypeptide-oligomer conjugates. Methods of synthesizing C-peptide polypeptide-oligomer conjugates and other pro-polypeptide-oligomer conjugates are also provided.

The present invention relates to a method of preparing insulin from proinsulin comprising converting high-concentration proinsulin into insulin by enzymatic cleavage, a method of purifying insulin, and insulin prepared therefrom.

A pharmaceutical formulation comprises insulin having a variant insulin B-chain polypeptide containing an ortho-monofluoro-Phenylalanine substitution at position B24 in combination with a substitution of an amino acid containing an acidic side chain at position B10, allowing the insulin to be present at a concentration of between 0.6 mM and 3.0 mM. The formulation may optionally be devoid of zinc. Amino-acid substitutions at one or more of positions B3, B28, and B29 may additionally be present. The variant B-chain polypeptide may be a portion of a proinsulin analogue or single-chain insulin analogue. The insulin analogue may be an analogue of a mammalian insulin, such as human insulin. A method of lowering the blood sugar of a patient comprises administering a physiologically effective amount of the insulin analogue or a physiologically acceptable salt thereof to the patient.

The present invention relates to human insulin expression plasmids and a method for producing insulin using the same. The plasmids comprise a sequence encoding a compound of the formula R—B—X-A, in which R is a leader peptide of the formula of Met-Thr-Met-Ile-Thr-Y (SEQ ID NO: 36), in which Y is one selected from lysine, arginine, a peptide containing lysine as an amino acid at its C-terminal, or a peptide containing arginine as an amino acid at its C-terminal; B is human insulin B-chain or analogue thereof; X is a peptide connecting B with A; and A is human insulin A-chain or analogue thereof. The method for preparing insulin using the plasmids according to the present invention converts the proinsulin fusion protein into human insulin in a single enzymatic cleavage process and minimizes the generation of by-products after the enzymatic cleavage, thereby producing insulin at a high yield. Therefore, the plasmids according to the present invention and the method for preparing insulin using the same can be usefully applied to the industrial mass-production of human insulin.

Methods to determine the concentrations in a sample of proinsulin and of molecules comprising C-peptide are disclosed. The concentrations are determined within the same reaction compartment and using only one capture antibody, specific for an epitope within C-peptide. Kits are disclosed for use in the disclosed methods. Methods to determine the concentrations in a sample of n analytes from a group of N analytes and the total concentration of the N analytes are disclosed. N is 2 or more and n is any integer value from 1 to N. Each of the N analytes has at least one common portion present in each other analyte, and the n analytes have each a distinct portion not present in any other of the N analytes. The concentrations are determined within the same reaction compartment using only one capture antibody which is specific for an epitope within the common portion.