IL-6 receptor IL-6 direct fusion protein

Abstract

The present invention intends to provide an IL-6R.IL-6 fusion protein and the like in which IL-6R and IL-6 are directly linked without a linker. The IL-6 receptor.IL-6 fusion protein of the present invention has a structure in which one amino acid residue constituting IL-6 receptor and one amino acid residue constituting IL-6 are directly bonded.

Description

[0001] The present invention relates to a fusion protein composed of an interleukin-6 receptor (hereinafter referred to as IL-6R) and an interleukin-6 (hereinafter referred to as IL-6) directly linked without a linker sequence; a gene for coding for the fusion protein; a host transformed by an expression vector containing the gene; a method of cultivating the host; a process for purifying the fusion protein derived from the culture of the host; a novel ex vivo amplifier containing the fusion protein for hematopoietic stem cells; and a novel blood platelet-proliferating agent.BACKGROUND TECHNIQUE[0002] IL-6, IL-11, ciliary neurotropic factors, leukemia inhibitory factors, oncostatin-M, and cardiotropin-1 belonging to IL-6 type cytokines are known to transmit signals through a receptor complex containing at least one signal-transmitting protein gp130. For example, IL-6 links to IL-6R, and the resulting IL-6.cndot.IL-6R complex contains gp130.[0003] The hematopoiesis system is one of t...

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Benefits of technology

0112] The fusion protein 333A.DELTA.A of the present invention described in Example 5 and purified by the method described in Example 7 (hereinafter referred to as FP6) was used in the experiment below.

0123] CD34-positive cells were isolated and purified from 20 mL of human umbilical blood according to the method disclosed in Japanese Patent Application No. 9-325847. The obtained 500 cells were cultivated with 1.2% methylcellulose (Shin-Etsu Kagaku K. K.), 30% bovine serum albumin (Hyclone Laboratories Inc.), 1% bovine serum albumin (hereinafter referred to as BSA) (Sigma Co.), 0.05 mM 2-mercaptoethanol (Sigma Co.), and SCF (stem cell factor) (100 ng/mL) under any of the conditions of (1) combination of IL-6 (100 ng/mL) and IL-6R (100 ng/mL), (2) combination of IL-6 (100 ng/mL) and IL-6R (200 ng/mL), (3) FP6 (300 ng/mL), and (4) FP6 (600 ng/mL), with dispensation of 1 mL fractions of .alpha.-MEM (Flow Co.) on a 35-mm plastic cultivation plate (Nunc Co.) for suspension cultivation at 37.degree. C., 5% CO.sub.2, and humidity 100%.

0124] Two weeks later, the colonies were identified by obse

Problems solved by technology

These findings are consistent with the fact that IL-6 administered to a mouse increases significantly the number of blood platelets and the IL-6 administered to a human body is limited in its effect.

In development of a fusion protein as a periodically dosed medicine, high possibility of the aforementioned immune reaction is a problem, since the linker is not contained in the proteins and irrelevant to them, and has an independent steric structure.

An excessive amount of methanol is toxic to the yeast, whereas the deficiency of methanol suppresses the function of the promoter sequence of the alcohol oxidase gene.

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