High efficiency finished purification method for colibacillus expression recombinant human interleukin-11

Abstract

The invention relates to an elaborate purification method of recombinant human interleukin 11 (rhIL-11). Fusion protein is desalinated by utilizing a dextran gel G-25 desalination column, the desalinated liquid is collected to carry out the cascade sample loading of negative and positive ion exchange chromatographic columns, after the sample loading is finished, the desalinated liquid is eluted by the specific Gly-NaOH buffer solution, and the collected main eluted protein solution is desalinated by the dextran gel G-25 desalination column, so that a high-purity protein sample is obtained. The method is suitable for large-scale production and is characterized in convenient operation, short production cycle, high purity of products, stable technique, etc.

Description

technical field [0001] The invention relates to the production of genetic engineering drugs by using recombinant DNA technology, in particular to a high-efficiency purification method for recombinant human interleukin-11 (rhIL-11) expressed by Escherichia coli, and belongs to the technical field of medicine. Background technique [0002] Interleukin-11 (IL-11) is an important cytokine in the human body. It can cooperate with other cytokines to stimulate the growth of human granulocytes, erythrocytes, and megakaryocyte precursor cells, induce the maturation of megakaryocytes, and promote platelet production. Natural human IL-11 consists of 178 amino acids, contains a lot of basic amino acids such as: proline, leucine, etc., does not contain cysteine, lacks disulfide bonds, isoelectric point is 11.7, and has a stable structure. [0003] In November 1997, rhIL-11 produced by the Genetic Institute of the United States was approved by the FDA for the treatment of regenerative thr...

AI Technical Summary

Problems solved by technology

In 1998, the Genetic Institute applied for a production method patent US5760189, which disclosed a method for highly expressing recombinant human interleukin-11 with a fusion protein. Greatly increased production costs, and at the same time caused great difficulties for downstream purification due to the introduction of heterologous proteins

[0004] Aiming at the deficiencies of the above-mentioned technologies, the patent CN1114617C discloses a method for cutting fusion proteins with hydroxylamine hydrochloride, thereby solving the above-mentioned problems, but for the fine purification of rhIL-11, this method The fusion protein hydroxylamine cleavage solution is adopted to be dialyzed, then through separate agarose gel (CM-Sepharose) column chromatography, and then carried out linear gradient elution with 0-1mol / LNaCl, there are following defects in this method: Dialysis removes hydroxylamine hydrochloride The time is too long, the processing volume is small, and it is not suitable for large-scale production; in addition, domestic literature reports have adopted the method of affinity chromatography. Although this method has strong specificity, the carrier is expensive, the mechanical strength is low, and the preparation of the ligand is difficult. There is the problem of high cost; there is also the method of molecular sieve chromatography, which can obtain high-purity samples, but the column efficiency requirements are strict, and the sample flow rate and sample volume are also limited, which is also not suitable. Expand production

[0005] In addition, for the purification of proteins containing high salt and high impurities similar to those after hydroxylamine cleavage, according to relevant reports, most of them use ultrafiltration to remove hydroxylamine hydrochloride and desalt, and then use separate agar Sugar gel (CM-Sepharose) column chromatography controls the elution component to obtain the target protein by controlling the ionic strength of the eluent. The disadvantage of this treatment is: the ultrafiltration method causes the adsorption loss of the target protein, and the desalination solution is directly applied The CM-Sepharose column will cause a large amount of impurity proteins to be adsorbed on the column, which affects the purity and yield of the product

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