Method for expressing and purifying human recombinant interleukin-3

Abstract

The invention discloses a Pichia pastoris transformant capable of expressing human recombinant interleukin-3 (rhIL-3) with high efficiency and a purification method thereof. In the method, the eukaryotic host is pichia pastorisX-33. The purification method comprises the following steps: cloning a human IL-3 gene; establishing a eukaryotic expression vector, and transforming the eukaryotic expression vector into the eukaryotic yeast host; obtaining a yeast transformant for high-level secretory expression by screening, wherein the IL-3 expressed by the yeasts are available in a glycosylated mode and a non-glycosylated module; and performing amplified culture by using a shake flask, and subjecting the supernate of the culture solution to dialysis, nickel affinity purification and further purification by diethylaminoethanol (DEAE) anion column. The purified product is subjected to mass spectrometric identification and analysis, and the result of the mass spectometric identification and analysis indicates that the expressed IL-3 is modified by different glycosyls and that the IL-3 has an his*6 tag and a C-MYC tag and is easy for purification and detection of expression product. In the invention, different from the conventional method using a prokaryotic host to express the rhIL-3, the method for expressing a large amount of rhIL-3 by using a Pichia pastoris expression system is adopted for the first time, quick purification is realized by using a His-tag protein, the purified rhIL-3 is high-activity rhIL-3 protein which is glycosylated to different extents and of which the molecular weight is 19kDa and 22kDa. The method ensures that the high-activity rhIL-3 recombinant protein is obtained quickly.

Description

Technical field [0001] The present invention relates to the application of recombinant DNA technology to produce genetically engineered protein drugs, in particular to an efficient method for expression and purification of human recombinant interleukin-3 (rhIL-3). Background technique [0002] Interleukin 3 (IL-3): also known as multipotent colony stimulating factor (multi-csf), mainly produced by activated cd4+ t cells. Its main function is to promote the directed differentiation and proliferation of pluripotent hematopoietic stem cells in the bone marrow, and to produce various types of blood cells. Because it is not produced in bone marrow stromal cells, it is not often associated with hematopoiesis. It is mainly involved in the body's defense function and is the inflammatory cytokine produced in response. In addition, il-3 can also regulate the growth, differentiation and related gene expression of a variety of mature cells, such as c-myc and il-2r α genes. The molecular we...

AI Technical Summary

Problems solved by technology

The expression product of Escherichia coli usually exists in the form of inclusion bodies, and the active form of IL-3 can only be obtained through complex operations of denaturation and renaturation, thereby reducing the yield of active protein; in addition, prokaryotic hosts express especially in Escherichia coli , because of the toxicity caused by the presence of LPS, it is often necessary to analyze and determine the toxicity of the expressed purified product; finally, to obtain a high-purity protein often requires multi-step purification operations, the more purification steps, the higher the protein yield. will be lower and more likely to lead to the inactivation of the target product

Although, insect cells can also express IL-3 protein, but its cost is high and the yield is very low

Therefore, the rhIL-3 recombinant protein currently on the market is extremely expensive

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