37 results about "Protein refolding" patented technology

A bioreactor that combines the steps of recombinant expression and separation of a biological product by binding the secreted biological product with a resin, discarding the nutrient medium and eluting the biological product as a concentrated solution, eliminating the steps of sterile filtration and volume reduction. The method also allows loading of resin for column-purification, eliminating all steps of perfusion process and maintaining a sink condition of a toxic product in nutrient medium to optimize productivity of host cells. The instant invention also allows harvesting of solubilized inclusion bodies after the cells have been lysed and refolding of proteins inside the bioreactor.

The present invention is a process for refolding and renaturing human serum albumin protein to substantially native conformation by the addition of a human serum albumin refolding ligand to a solution containing the protein under conditions conducive to refolding of the protein.

The present invention discloses improved methods of disaggregating protein aggregates, and refolding denatured proteins, using high pressure. In particular, the present invention provides for the use of agitation, high temperature, "stepped" depressurization, dialysis and dilution under pressure to increase the speed and extent of aggregate dissolution and protein refolding.

This invention relates to the use of ionic liquids comprising a cation with at least one electron donor region and one positively charged electrostatic region which are spatially distinct from each other for protein refolding and a method for refolding proteins using said ionic liquids.

A novel approach is described for reversing aggregation and increasing refolding by application of hydrostatic pressure. A protein of interest in an aggregated, or inclusion body, or other non-native or inactive state is subjected to high hydrostatic pressure. This treatment denatures the protein to states (or conformations) competant for refolding and results in increased formation of native protein once pressure is released. The technique can facilitate conversion non-native proteins, including inclusion bodies and aggregates to native proteins without addition of chaotropic agents, changes in buffer, or large-scale dilution of reagents required for traditional refolding methods.

The present invention discloses improved methods of disaggregating protein aggregates, and refolding denatured proteins, using high pressure. In particular, the present invention provides for the use of agitation, high temperature, “stepped” depressurization, dialysis and dilution under pressure to increase the speed and extent of aggregate dissolution and protein refolding.

The present invention relates to a method for the manufacture and purification of recombinant trypsinogen and trypsin in E. coli and yeast, using high yield expression vectors with and without secretion leader sequences. The invention further relates to an improved method and apparatus for carrying out protein refolding specifically useful for processing trypsinogen that has accumulated intracellularly in the form of inclusion bodies.

The invention refers to a protein refolding method using metal ion chelate affinity chromatography column as solid phase carrier. The invention uses the specificity affinity relation between rFGF with His or SUMO labels and metal ion chelate affinity chromatography column, attaches the denatured and reduced His-rFGF or SUMO-rFGF to the metal ion chelate affinity chromatography column, replaces denatured buffer solution with refolding buffer solution by gradually changing the composition of mobile phase, so as to prevent the refolding protein folding intermediates from gathering together, and obtain highly effective protein refold while finishing the purification of target protein. Compared to the traditional refolding by dialysis, the method not only can greatly reduce the time needed for protein refolding, but also can make the denatured His-rFGF or SUMO-rFGF obtain refolding protein with high purity and activity in the high concentration(1-10mg / mL), further providing a new process for accomplishing highly effective protein refolding and purifying.

The present invention provides a method for producing a protein which has a restored native higher-order structure by bringing a protein which has lost its native higher-order structure into contact at pH 6.5 to 9.0 with a 1 to 3% aqueous solution of a specific surfactant, such as lauroylglutamic acid to obtain a solubilized solution of the protein; and then adding the solubilized solution to a buffer with pH 6.5 to 9.0 containing arginine or an arginine derivative at a concentration of 0.1 to 1.2 M to lower the concentration of the specific surfactant, such as lauroylglutamic acid, in the obtained mixture solution down to 0.02 to 0.275%. According to the present invention, it is possible to easily restore the native higher-order structure of a protein while smoothly removing the surfactant from the protein.

The present invention relates to an efficient and improved process for purifying a recombinant protein. The invention relates to the purification of tissue plasminogen activator (tPA), such as truncated human tPA, recombinantly produced in bacteria, for example in E. coli. The present invention provides a process that requires less refolding volume after solubilization of inclusion bodies isolated from cells expressing the recombinant tPA, without affecting the yield and purity of the tPA protein. The invention also provides optimum arginine concentrations for use during protein refolding and during ion exchange chromatography.

A refolding agent and refolding method which make it possible to produce high-purity proteins in high productivity. The refolding agent includes a phosphorus-containing compound (A) and an oxycarbonyl group-containing compound (B). The refolding method includes the step of treating the unfolded protein with the refolding agent. As the compound (A), there may be mentioned at least one species selected from inorganic phosphoric acids, alkyl phosphate esters, sugar phosphate esters, and salts of these, and as the compound (B), there may be mentioned at least one species selected from formic acid, acetic acid, propionic acid, lactic acid, tartaric acid, and salts of these.

The invention discloses a production process of a chicken beta interferon optimized gene and a recombinant chicken beta interferon. The process includes the steps of: S1. conducting codon optimization on published chicken beta interferon genes in NCBI according to the preference of Escherichia coli codon and performing artificial synthesis of the optimized chicken beta interferon gene; S2. designing primers: according to the codon optimized interferon gene, designing a pair of primers; S3. constructing a recombinant expression plasmid; S4. transforming and identifying the recombinant expression plasmid; and S5. carrying out induced expression of the recombinant expression plasmid in Escherichia coli; and S6. extracting the expression product and conducting protein refolding and purification: S61. extraction and treatment of an inclusion body; S62. inclusion body denaturation; S63. denaturation fluid renaturation; and S64. nickel column affinity purification. The process provided by the invention expresses and purifies the recombinant chicken beta interferon protein by means of prokaryotic expression, and detects the interferon's activity of inhibiting vesicular stomatitis virus proliferation by means of cytopathic effect inhibition, and the activity unit reaches 2.73*10<6>UI / mg.

The present invention provides a method for producing a protein which has a restored native higher-order structure by bringing a protein which has lost its native higher-order structure into contact at pH 6.5 to 9.0 with a 1 to 3% aqueous solution of a specific surfactant, such as lauroylglutamic acid to obtain a solubilized solution of the protein; and then adding the solubilized solution to a buffer with pH 6.5 to 9.0 containing arginine or an arginine derivative at a concentration of 0.1 to 1.2 M to lower the concentration of the specific surfactant, such as lauroylglutamic acid, in the obtained mixture solution down to 0.02 to 0.275%. According to the present invention, it is possible to easily restore the native higher-order structure of a protein while smoothly removing the surfactant from the protein.

An apparatus for refolding proteins and a method of using an apparatus for refolding proteins is provided in which a refolding process for proteins can be carried out for various proteins, efficiently and accurately, and without manual intervention. The construction comprises: a transport section on which either one or two or more vessels capable of holding a protein solution containing a protein to be refolded are placed, which transports the vessels along a predetermined closed transport path; an optical measurement section provided on the transport path, which measures the optical properties of the protein solution within the vessel; a pH measuring section provided on the transport path, which measures the pH of the protein solution within the vessel; and a reagent supply section provided on the transport path, capable of supplying each of a plurality of types of reagent including reagents for adjusting the pH of the protein solution within the vessel, wherein the reagent supply section performs protein refolding by adjusting the pH of the protein solution based on the measurement results of the optical measurement section, the measurement results of the pH measuring section, and a predetermined pH pattern.

The invention provides methods and systems for production of recombinant protein, and particularly, for production of recombinant protein from inclusion bodies. For example, in one aspect, the method comprises providing a protein preparation comprising inclusion bodies, preparing an inclusion body dispersion, and exposing the protein preparation to high pressure in a pressure vessel, to disaggregate and refold the inclusion body protein.

The invention relates to an analysis method of protein refolding in electrical contraction polymer. The invention utilizes the crosslink network of electrical contraction polymer to insulate modifiedprotein molecule and removes modifier and contracts polymer at electric field, to loosen the polymer packing modified protein, therefore, modifier protein can fold without interfered and coiled by other proteins, and without the generation of inclusion body. The inventive analysis method builds a model of external molecule mate, for protein folding dynamic research.

The invention discloses a novel application of fibrinogen-420 and its active domain (alpha EC domain), and a separate alpha EC domain protein has the same or similar function with fibrinogen-420. Fibrinogen-420 and its active domain can be widely used in inhibiting protein aggregation, helping protein refolding, drugs which can prevent and / or treat protein conformation disease, detecting denatured protein in quality control and protect protein from denaturation.

The present invention provides a method for producing a refolded protein, the method comprising a step for mixing, inside a micro mixer, a buffer and a solubilization solution for a protein that has lost higher order structure or that has become insoluble.

It is an object of the present invention to provide: a protein refolding column filler, which is effective for the refolding, namely, the activation of the function, of an inactive protein with an as yet unformed higher order structure produced in Escherichia coli or the like, or a protein whose conformation has been changed due to a certain cause and which has become inactivated; and a column filled with the aforementioned column filler. The present invention provides a protein refolding column filler, which comprises zeolite with BEA structure (Zeolite Beta) that is granulated into a particle state.