54 results about "Insulin Precursor" patented technology

Methylotrophic recombinant yeast strain producing human insulin precursor, the strain comprising, in its genome, a copy of a first DNA construction and a second DNA construction, wherein the constructions are capably of directing the expression and secretion of human insulin precursor of the formula B(1-30)-Y1-Y2-A(1-21), wherein Y1 is lysine or arginine; Y2 is lysine or arginine; B(1-30) is the B peptide of the human insulin; and B(1-21) is the A peptide of human insulin, and wherein the strain is yeast Pichia Pastoris. DNA constructions and method for obtaining the strain are also provided.

The invention discloses a purification and enzyme digestion transformation method of a recombinant human insulin precursor and belongs to the field of preparation of recombinant human insulin and analogues thereof. In the invention, fermentation supernatant of a secretorily expressed recombinant human insulin precursor is purified by using a cation exchange chromatography column, two different washing solutions are used during washing of chromatography, the elution of target proteins is performed more quickly, the elution time is shortened, the precipitation of the target proteins is reduced and the volume of eluted samples is reduced; an eluting solution with pH 7.0 to 9.0 is used during elution, phase change operation does not need to be performed to eluted products, the eluted products are directly added into trypsin for enzyme digestion transformation and the transformation efficiency is high. The method disclosed by the invention is simple to operate, the primary purification and phase change operation of the fermentation supernatant are completed through one step of chromatography purification, the purity of the samples can be improved from 14 percent to 95 percent, the purified samples are directly subjected to enzyme digestion, the enzyme digestion transformation efficiency is above 95 percent, the production process is simplified and the cost is saved.

A method for supplementing and optimizing feed during fermentation of insulin precursor is carried out by supplementing methanol to determine its fluid acceleration ratio, computing while adjusting for specific growth speed ration by difference of final fermented strain density and real-time strain density and fermentation period and controlling growth speed ratio. It has more protein concentration.

The invention relates to the field of insulin production methods and discloses a recombinant insulin and insulin analogue precursor purification method. The invention solves the problems that conventional chromatographic packing cannot tolerate high-salinity sample loading, the amount of used organic reagent is great, the cost is high and the product purity is not high in the purification process of recombinant expressed insulin precursors and insulin analogue precursors. The invention adopts the technical scheme that the method comprises the steps of performing pH regulation and centrifugation to centrifuged fermentation supernatant, then directly loading a sample and performing adsorption, separation, purification and elution through a chromatographic column prepared by using any packing of Capto S, Capto MMC, Uni PMM S and Uni MSP to finally obtain high-purity recombinant insulin and insulin analogue precursors. Compared with the existing purification method, the recombinant insulin and insulin analogue precursor purification method has the advantages that the operation is simple, the yield is high, the spent time is short, the environmental influence is small, and the product production cost of the existing insulin and insulin analogues can be greatly reduced.

The invention provides a novel process for genetic engineering preparation of insulin and insulin analogs, which comprises enzyme cutting insulin precursor with parenzyme, then isolating insulin, wherein the insulin precursor comprises the following elements from amino end to carboxyl end, (1) spacing peptide element with formula (Xaa)mZ, (2) insulin B chain element, (3) joint peptide element with formula (Xaa)nZ, and (4) insulin A chain element.

The invention relates to a human insulin/analogue conjugate with a continuous blood sugar reduction function and a high rate of combination with a receptor, and discloses a method for preparing a reconstructed human insulin/analogue conjugate by reconstructing a human insulin precursor by utilizing a methylotrophic yeast expression, performing purification, enzyme digestion, chromatography or synthesis to prepare a reconstructed human insulin and a DesB30 analogue thereof and coupling a side chain amino group of a 29th-bit lysine of a B chain of the reconstructed human insulin precursor and a polyethyleneglycol acetamide bond. The prepared reconstructed human insulin conjugate has the characteristics of high rate of combination with the insulin receptor, remarkable continuous blood sugar reduction effects and long in-vivo half-life period, and is applied to the treatment of I-type and II-type diabetes.

The present invention relates to a chimeric protein containing an intramolecular chaperone (IMC) like sequence linked to a target protein, preferably an insulin precursor. The present invention also relates to a process for obtaining a correctly folded insulin-precursor-containing chimeric protein, comprising, inter alia, contacting an incorrectly folded chimeric protein containing an IMC like sequence linked to an insulin precursor with at least one chaotropic auxiliary agent. The present invention further relates to an assay for screening an amino acid sequence for the ability to improve folding of an insulin precursor using a chimeric protein containing an IMC like sequence linked to an insulin precursor.

A preferred way of converting insulin precursors into insulin compounds is to perform an enzymatic peptide cleavage in an aqueous medium and, thereafter, without removal of the intermediate product formed, to add an amino acid ester or a peptide ester and an organic solvent so that the desired coupling takes place.

The invention discloses a preparation method of insulin detemir or an insulin detemir analogue and belongs to the field of preparation of insulin analogues. According to the preparation method, threonine-free 30th-site human double-chain insulin precursor desB30 protein of a B chain is taken as a raw material, epsilon-amino of B29th-site lysine is selectively acylated by active ester through one step in the alkaline environment with pH ranging from 8.0 to 11 under the condition that N-terminal alpha-amino of double chains of the desB30 protein is not protected, and the insulin detemir or the insulin detemir analogue is generated efficiently. According to the preparation method of the insulin detemir or the insulin detemir analogue, parameters such as the pH value, the raw material ratio, the acylation time and the like of the acylation reaction of the insulin precursor are optimized, the yield of the insulin detemir or the insulin detemir analogue is remarkably increased, the preparation method has the advantages of high acylation efficiency, a few by-products, short acylation reaction time, a few acylation steps, simple process, low production cost and the like, and one simple and efficient method is provided for large-scale industrial preparation of the insulin detemir or the insulin detemir analogue through one-step acylation.

The invention discloses a preparation method of recombinant human insulin. The preparation method comprises the following steps of: performing an enzyme digestion reaction on a recombinant human insulin precursor in a mixed solution of an organic solution and water to obtain a human insulin solution containing desB30; centrifuging or suction filtrating the human insulin solution containing desB30 to obtain water-containing powder of desB30 human insulin; performing a transpeptidation reaction on the water-containing powder of desB30 human insulin to obtain human insulin ester, wherein the final concentration of the desB30 human insulin in a transpeptidation reaction system is 12.1mM-33mM, the mol ratio of the desB30 human insulin to threonine ester or a derivative thereof is 1:(15-150), and the mass ratio of the desB30 human insulin to trypsin is (10-80):1; degreasing the recombinant human insulin ester to obtain the recombinant human insulin. The method is suitable for industrial production of converting yeast-expressed proinsulin into the human insulin, high in yield, simple to operation and low in cost.

The present invention relates to a chimeric protein containing an intramolecular chaperone (IMC) like sequence linked to a target protein, preferably an insulin precursor. The present invention also relates to a process for obtaining a correctly folded insulin-precursor-contain chimeric protein, comprising, inter alia, contacting an incorrectly folded chimeric protein containing an IMC like sequence linked to an insulin precursor with at least one chaotropic auxiliary agent. The present invention further relates to an assay for screening an amino acid sequence for the ability to improve folding of an insulin precursor using a chimeric protein containing an IMC like sequence linked to an insulin precursor.

The invention relates to a preparation method of insulin. The method which comprises two steps of chromatography purification can improve the enzymatic digestion efficiency, and the controllability in an enzymatic digestion process, so a complete reaction of trypsin and carboxypeptidase B is realized, and insulin precursors not reacted with the carboxypeptidase B can be thoroughly removed, so the purity and the quality of final insulin products are improved, the purity of the final insulin products can reach above 99.7%, and the recovery rate is high, thereby industrialized production can be carried out.

The invention discloses an enzyme digestion conversion method of a recombinant human insulin precursor. The enzyme digestion conversion method comprises the following steps: (1) adding a primarily purified recombinant human insulin precursor solution into a buffer solution, and adjusting a concentration and a pH value to obtain an enzyme digestion reaction substrate mixed solution; and (2) adding recombinant lysyl endonuclease into the enzyme digestion reaction substrate mixed solution to carry out enzyme digestion conversion reaction on the recombinant human insulin precursor. The recombinant lysyl endonuclease is selected as a tool enzyme, and compared with common recombinant trypsin, the recombinant lysyl peptide endonuclease has advantages that less enzyme is needed by unit precursor protein, enzyme digestion site is single, enzyme digestion process is more efficient, and enzyme digestion conversion efficiency of a human insulin precursor reaches 95% or above; when enzyme digestion is amplified, worry about excessive enzyme digestion is avoided, and purity of target protein obtained after enzyme digestion is higher; the optimum pH range of enzyme digestion of the recombinant lysyl endonuclease is farther from pI of the protein before and after enzyme digestion, so that stability of the target protein is facilitated, and tolerance range to a salt concentration in the enzyme digestion environment is wider.

The invention provides a genetically engineered bacterium for expressing an insulin precursor. The genetically engineered bacterium is an engineered bacterium in which an expression vector of an insulin precursor gene is integrated in pichia pastoris, and the nucleotide sequence of the insulin precursor gene is shown as SEQ ID NO.1 in a sequence table. The invention also provides a preparation method of the insulin precursor. The strain yield of the genetically engineered bacterium fermented at a shake flask level is higher than the strain yield reported in the prior art. Through combination of the genetically engineered bacterium and the preparation method of the insulin precursor, the yield of the insulin precursor obtained by fermentation tank-level fermentation is remarkably increased.Besides, a culture medium used in the preparation method of the insulin precursor is relatively low in cost, and the risk of animal-derived virus pollution is avoided, so that cost of the preparationmethod is remarkably reduced.

The invention belongs to the technical field of biopharmaceuticals, and discloses a fermentation medium of pichia pastoris for improving the expression of insulin precursor and a method for fermentedproduction of the insulin precursor. The fermentation medium of pichia pastoris in the present invention adopts a phosphate buffer system, supplemented by inorganic salts such as ammonium sulfate andferrous sulfate, as well as nitrogen sources such as urea and ammonium dihydrogen phosphate. The fermentation medium has definite compositions with stable sources and does not require fed-batch of nitrogen sources in the induction stage. According to the method for fermented production of the insulin precursor, a glycerol/methanol mixed solution is adopted for induction of culture after initial culture and glycerol fed-batch culture. A certain proportion of glycerol is fed by batches in the induction stage, so as to accelerate the growth of microorganisms. As time increases, the mixing ratio of glycerol gradually decreases while methanol gradually increases, and a large number of microorganisms enter the high-speed expression period in a short time, so that a target product can obtain a higher level of expression, greatly shortening the culture time and avoiding influence on product quality caused by excessive by-products.

The invention discloses a method for preparing insulin or an insulin derivative precursor. The method comprises the following steps that S1, protease is added to fermentation broth of insulin precursor protein; S2, the pH value and temperature of the solution treated in S1 are adjusted, so that protease is subjected to digestion, and a solution containing the insulin or insulin derivative precursor is obtained after digestion is carried out. The method for preparing the insulin or insulin derivative precursor can further comprises the step that S3, the solution containing the insulin or insulin derivative precursor obtained after digestion is carried out is subjected to isoelectric precipitation recycling operation, and the higher-purity insulin or insulin derivative precursor is obtained.Compared with the prior art, according to the scheme, the method for preparing the insulin or insulin derivative precursor is easy and convenient to operate, low in equipment investment and high in technological process yield and has good industrial application prospects.