CN1114617C

CN1114617C - Process for preparing recombined human interleukin-11 by use of hydroxylamine to cut fusion protein

Info

Publication number: CN1114617C
Application number: CN 99112337
Authority: CN
Inventors: 王革
Original assignee: Individual
Current assignee: Individual
Priority date: 1999-07-19
Filing date: 1999-07-19
Publication date: 2003-07-16
Anticipated expiration: 2019-07-19
Also published as: CN1280984A

Abstract

The present invention discloses a method for preparing IL-11 by using a cheap chemical reagent to cut interleukin 11 fusion protein. The fusion protein comprises escherichia coli thioredoxin and IL-11, wherein a specific cutting site of Asn-Gly of hydroxylamine is led in the connection point of the escherichia coli thioredoxin and IL-11, so an Asn-Gly peptide bond can be uniquely cut off by hydroxylamine to release integral IL-11 after the fusion protein is effectively expressed by escherichia coli in use, and integral recombination IL-11 with high purity and high specific activity can be manufactured after purification in some steps.

Description

Method with cutting fusing protein with hydroxylamine prepares recombination human interleukin 11 using

The invention belongs to the recombinant protein drug in the field of medicaments.

Interleukin-11 (Interleukin11 is called for short IL-11) is a class important cytokine of human endocrine, and it acts on the original hemopoietic stem cell in the medullary cell, promotes megakaryocytic maturation, and the platelet increasing number promotes blood platelet regenerating.Recombinant interleukin 11 was gone on the market by drugs approved by FDA in November, 1997, became the treatment cancer patient through unique medicine of putting, platelet count reduces after the chemotherapy.

At present, producing IL-11 with gene engineering method is unique valid approach.But the complete IL-11 of reorganization directly dissociates very big difficulty is arranged when expressing in intestinal bacteria, so that it is impossible, this is because complete IL-11cDNA 5 end GC content are abundant especially, in especially preceding 8 amino acid 6 proline(Pro) is arranged, and has seriously hindered the direct expression of complete IL-11.For the IL-11 of The expressed can only promptly add one section leading peptide with the method for fusion rotein before the IL-11N end, the formation fusion rotein so that IL-11 efficiently expressed, and then cuts away the leading peptide in the fusion rotein, produces complete IL-11.The general now method that adopts is to come cleavage of fusion proteins to produce IL-11 with single-minded proteolytic enzyme intestines peptide kinases (enterokinase), but this kind of enzyme blanking method has produced cutting and has cost an arm and a leg with enzyme and serious problems such as heterologous protein pollution, make reorganization IL-11 cost high, purification difficult.

The objective of the invention is to efficiently express the fusion rotein of the complete IL-11 of reorganization with escherichia expression system, and insert the chemical reagent cleavage site at the leading peptide of fusion rotein and the combining site between the IL-11, so that production cost significantly reduces, purifying process is simplified, and the biologic activity of IL-11 remains unchanged simultaneously.

Principle of the present invention: azanol can cut to specificity the Asn-Gly peptide bond in albumen and the polypeptide as cheap albumen cutting chemical reagent.

We find in experiment, do not contain azanol cleavage site Asn-Gly peptide bond in the interleukin 11 polypeptide, and its N end is glycine Gly just, we are the N end introducing azanol cleavage site Asn-Gly (AACGGG) of IL-11 at the leading peptide of fusion rotein and the junction of IL-11 like this, behind fusion rotein with escherichia coli high-level expression IL-11, cut with azanol again, cut out the N end just and be the complete IL-11 of Gly, and have natural biologic activity.

The engineering strain that after above-mentioned transformation, transforms, behind fermentation expression, through microorganism collection → bacterial cell disruption → centrifugation → purification steps such as anionresin layer folding → azanol cutting → cationic exchange layer folding, can prepare purity greater than 95%, specific activity reaches 8 * 10 ⁶The complete interleukin 11 of IU/mg.

Most preferred embodiment:

In the clone's process that makes up the IL-11 fusion rotein, place escherichia coli thioredoxin (thioredoxin) as leading peptide in the front of IL-11, introduce azanol cleavage site Asn-Gly between thioredoxin and IL-11, wherein Gly not only is the amino acid in this site but also be the N terminal amino acid of IL-11.Above-mentioned improved IL-11 fusion rotein cDNA is placed among the expression vector pThioA, be transformed into escherichia coli DH5a, can efficiently express generation IL-11 fusion rotein with IPTG inductive mode, expression level reaches 30%, the fusion rotein of thioredoxin guiding simultaneously is present in the colibacillary cytoplasm with solvable state, and the tool natural radioactivity.This advantage can be removed the required problems such as sex change, renaturation and isomer separation of albumen of inclusion body form from.After with the azanol cutting, produce complete IL-11.Purifying process is simplified like this, and production cost reduces greatly.

Expression and preparation method are as follows: picking one single bacterium colony on the bacterial classification flat board, be inoculated in that (LBA: every liter contains the 10g peptone in the 2ml LBA substratum, the 5g yeast extract paste, 5gNaCl, 60mg penbritin, PH7.0), 37 degree overnight incubation are as seed, and the inoculum size by 1% is inoculated among the 50ml LBA, when 37 degree grow to O.D600=0.5, added IPTG to 1mM abduction delivering 6 hours, centrifugal results thalline.Thalline is suspended in 50mMPB (Na ₂HPO4+NaH ₂PO4, pH8.0) in the damping fluid, the ultrasonication thalline, 20000g * 20 are minute centrifugal.Get supernatant, supernatant is crossed anion column DEAE-Sepharose chromatography purification, uses 20mMPB, and the PH8.0 buffer solution elution is collected IL-11 fusion rotein enrichment peak.Add azanol again to 2M, transfer PH7-9, cutting is 2-20 hour under the 37-45 degree, discharges complete IL-11.Be further purified with cation seperation column CM-Sepharose again after the dialysis, use 0-1M NaCl, 20mM PB, the PH8.0 linear gradient elution washes the active peak of IL-11 at concentration place, the 0.25mM NaCl left and right sides.The SDS-PAGE electrophoretic analysis shows that the IL-11 purity that obtains is surveyed slip-knot and really reached 8 * 10 for specific activity greater than 95% ⁶IU/mg.By aforesaid method, can obtain high purity, the complete recombinant interleukin 11 of high specific acitivity.

The invention provides and a kind ofly with low cost express and cut the method for preparing IL-11 with the fusion rotein mode.It is compared with the production technique of the U.S. Genetics Institute of unique production IL-11 in the world has following advantage: 1, the fusion rotein cutting reagent is cheap chemical reagent-azanol, and the technology of G.I. company needs come cleavage of fusion proteins with expensive enterokinase.2, the azanol of this process using is the small molecules inorganic substance, does not bring heterologous protein to pollute.And exist the heterologous protein pollution problem of the enterokinase of fusion rotein leading peptide and adding in the technology of G.I. company.3, determined this technology purifying convenient and with low cost by above-mentioned two characteristics.Above-mentioned advantage makes the present invention have tangible technological innovation and technology advance, is more suitable in suitability for industrialized production and economical and practical.

Claims

1, recombinant interleukin 11 fusion roteins of the special cutting of available azanol, l-asparagine Asn is introduced in the preceding design of N end glycine Gly that it is characterized in that IL-11 in this fusion rotein, so just introduced an azanol specificity cleavage site Asn-Gly, thereby such fusion rotein of escherichia coli expression can be cut to discharge complete reorganization IL-11 with azanol at the N of IL-11 end.

2, in fusion rotein as claimed in claim 1, leading peptide is escherichia coli thioredoxin thioredoxin, it is characterized in that its bootable IL-11 that merges later is present in the colibacillary cytoplasm with solvable state, and make it to have natural biological and learn active.

3, azanol as claimed in claim 1 cuts the IL-11 fusion rotein, it is characterized in that adopting the cutting condition of optimization, and azanol concentration is 0.5～4M, and reaction pH is 7.5～9.5, temperature of reaction 37～45 degree, 2～20 hours reaction times.