Soluble CD105 protein eukaryotic expression and purification method

Abstract

The invention belongs to the technical field of mutation or genetic engineering for introducing foreign genetic materials by using a vector, and in particular relates to a soluble CD105 protein eukaryotic expression and purification method. The method comprises the following steps: A. selecting an extracellular region fragment (E26-G586) capable of expressing CD105 protein, wherein signal peptideof the CD105 protein is selected as a front-end signal peptide of the fragment, and respectively adding a Kozak sequence to the front end of the signal peptide and K_6*His to the tail end of the fragment so as to obtain a CD105 expression cassette; B. inserting the CD105 expression cassette into pcDNA3.4 plasmid to construct the eukaryotic expression vector pcDNA3.4-sCD105; C. performing transfection and expression by using HEK293F cells; purifying the target protein by using a two-step purification method. The method utilizes eukaryotic cell expression and purification to obtain the CD105 protein with a high purity (more than 95%), and the acquisition rate is as high as 4.17 mg / L.

Description

technical field [0001] The invention belongs to the technical field of mutation or genetic engineering for introducing foreign genetic material by using a carrier, and in particular relates to a eukaryotic expression and purification method of soluble CD105 protein. Background technique [0002] As early as 1985, Quackenbush and others found 39 groups of completely different antigens on the surface of the non-B and non-T acute lymphoblastic leukemia cell line HOON using the Mab obtained by hybridoma technology. Among them, the group IV membrane antigen is detected by Mab44G4, and is mainly located on the vascular EC of each tissue, that is, CD105 ("Identification of several cell surface proteins of non-T non-B lymphoblastic lenkemia by using monoclonal antibodies", Quackenbush E.J.et al , J. Immunol, No. 134, 1985, p. 1276, published December 31, 1985). In 1988, Gougos et al. further studied the membrane antigen and believed that it could become a new marker of EC and play ...

AI Technical Summary

Problems solved by technology

[0006] However, there are many eukaryotic expression methods, and a difference in one element or condition may lead to completely different final protein yields

The price of soluble CD105 protein expressed in eukaryotic currently sold is very expensive, and 1 mg of protein is close to 100,000 yuan, and the yield of soluble CD105 eukaryotic expression and purification methods reported so far is very low at 0.6 mg / L, and the purification process is complicated (“Structural and functional characterization of soluble endoglin receptor", B.V.Le et al, Biochemical&Biophysical Research Communications, Vol. 383, No. 4, 2009, pages 386-391, published on December 31, 2009)

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