A preparation method of recombinant human leukemia inhibitory factor
A technology of inhibitory factor and leukemia, applied in the field of medicine, can solve the problems of protein pollution, low renaturation efficiency, cumbersome process, etc.
Active Publication Date: 2019-10-29
BEIJING HAOGUYUANFANG BIOMEDICAL TECH CO LTD
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The LIF protein expressed by Samal BB et al. in Escherichia coli exists in the form of inclusion bodies, and after renaturation, the LIF protein was purified with reference to the method of the above-mentioned Hilton DJ et al. (Samal BB et al.Biochim Biophys Acta, 1995,1260 :27-34), but because the inclusion body needs a large amount of denaturant in the process of denaturation and renaturation, and the efficiency of renaturation is low, so this method is also not suitable for the large-scale preparation of LIF protein
In addition, some researchers have tried to express LIF protein in E. coli in the form of fusion protein. Proteins fused with LIF can usually promote the soluble expression of LIF protein (CN103981205, Jung AS et al.PLoS ONE, 2013, 8:e83781), but due to The protein expressed in the form of fusion needs subsequent digestion steps to obtain the untagged target protein. The process is cumbersome, and it is easy to introduce contamination from other proteins (such as enzymes used for digestion and protein tags expressed in fusion) , at the same time, because of the need to apply affinity chromatography in the purification process, the cost is often high
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[0022] 1. Preparation of E. coli Lysate
[0023] Tris-HCL, NaCl, and EDTA were mixed, and the concentrations of the three in the mixture reached 20 mM, 60 mM, and 1 mM, respectively, and then Triton X-100 was added to make the concentration reach 0.5%, to obtain a lysate for later use.
[0024] Inoculate Escherichia coli containing the expression vector of human leukemia inhibitory factor into LB medium, cultivate overnight at 37°C and 200 rpm, and then inoculate in freshly prepared LB medium at a volume ratio of 1:50, at 37°C and 200 Turn the shaking culture until the bacterial OD600nm detection result reaches 0.6, add IPTG with a final concentration of 0.5mM to induce, then reduce the temperature of the bacterial culture solution to 16°C, and change the bacterial culture temperature to 16°C at the same time, continue to shake at 200 rpm Bacteria were harvested after shaking for 20 hours.
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The invention relates to the field of medicines and particularly provides a method of preparation method for a human recombinant leukemia inhibitory factor by applying cell expression products of escherichia coli. In particular, the method includes the steps of: expressing leukemia inhibitory factor protein by applying the escherichia coli; performing low-temperature induction to achieve soluble expression of the leukemia inhibitory factor protein; and performing three-step purification, comprising cationic exchange chromatography, anionic exchange chromatography, and cationic exchange chromatography, to purify the leukemia inhibitory factor protein which has high purity and high activity. The method is easy to carry out, is low in cost and can be used for preparing the recombinant leukemia inhibitory factor in large scale.
Description
technical field [0001] The invention relates to the field of medicine, in particular to a method for preparing recombinant human leukemia inhibitory factor with high purity from Escherichia coli cell expression products. Background technique [0002] Leukemia inhibitory factor (LIF) consists of 180 amino acids and belongs to the IL-6 family. Its molecule contains 6 cysteines, which can form 3 pairs of disulfide bonds (cys12-cys134, cys18-cys131, cys60-cys163). Its receptors include two subunits, namely LIFR and gp130, both of which have an intracellular region and a transmembrane region. It is known that the downstream signaling pathways that can be activated by LIF include JAK / STAT3, PI3K / AKT, ERK1 / 2 and mTOR signaling It is known that LIF can promote the proliferation of stem cells and prevent their differentiation. At the same time, it has a significant impact on the functions of embryonic development, nervous system, immune and endocrine system, especially the prolifera...
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IPC IPC(8): C12P21/02C07K14/54C07K1/36C07K1/34C07K1/18
CPCC07K14/5415C12P21/02
Inventor 杜伯雨宋伟关欣李小璐吴帆郗雪艳
Owner BEIJING HAOGUYUANFANG BIOMEDICAL TECH CO LTD