Polypeptide of recombinant human bone morphogenetic protein-2

Abstract

The present invention relates to a field of biotechnology. It discloses a novel amino acid sequence of recombinant human bone morphogenetic protein-2 (rhBMP-2) and encoded nucleotide sequences thereof, and a method for preparing rhBMP-2. In the present invention, by further optimizing the nucleotide sequence of rhBMP-2, novel amino acid sequence of rhBMP-2 that can express good ectopic induced osteogenic activity and has good renaturation and purification effect is screened out. The engineered bacteria constructed by the present invention can induce the production of recombinant rhBMP-2 protein with an expression level of about 55%, and the produced target protein is more easily denatured and purified than the existing recombinant rhBMP-2, with better denaturation and purification effect and higher osteoinductive activity.

1 . A polypeptide of a recombinant human bone morphogenetic protein-2, wherein the polypeptide comprises the amino acid sequence SEQ ID NO: 1. 
     
     
         2 . The polypeptide according to  claim 1 , wherein the polypeptide comprises the amino acid sequence SEQ ID NO: 2. 
     
     
         3 . The polypeptide according to  claim 2 , wherein the polynucleotide encoding the amino acid sequence SEQ ID NO: 2 is SEQ ID NO: 3. 
     
     
         4 . The polypeptide according to  claim 2 , wherein the recombinant human bone morphogenetic protein-2 is prepared according to the following method and the method comprises the following steps:
 1) designing DNA sequences encoding recombinant human bone morphogenetic protein-2;   2) constructing an expression vector to transform  E. coli  host cells;   3) screening positive clones for culture and inducing the expression of the target protein;   4) renaturing and purifying the expression product to obtain the recombinant human bone morphogenetic protein-2;   wherein the DNA sequence is shown in SEQ ID NO: 3.   
     
     
         5 . The polypeptide according to  claim 4 , wherein a dilution method is used for renaturation in the step (4) and the final concentration of the protein in the renaturation buffer is controlled at 0.05-0.5 mg/ml. 
     
     
         6 . The polypeptide according to  claim 4 , wherein multi-step ion exchange chromatography is used for purification in step (4) and the steps are as follows:
 1) loading the renatured recombinant human bone morphogenetic protein-2 solution onto a well-balanced strong anion column, and rinsing with equilibration buffer A to reach the baseline after loading;   2) performing stepwise salt-gradient elution using the elution buffer A and collecting the main peak;   3) mixing the target peak solution collected from the strong anion column and loading onto a weak cation column, and rinsing with equilibration buffer B to reach the baseline after loading;   4) performing stepwise salt-gradient elution using the elution buffer B and collecting the main peak.   
     
     
         7 . The polypeptide according to  claim 6 , wherein the equilibration buffer A comprises 10-50 mM Tris-HCl, 1-5 M urea, 1%-10% mannitol, pH 8.5-8.9. 
     
     
         8 . The polypeptide according to  claim 6 , wherein the elution buffer A comprises 10-50 mM Tris-HCl, 1-5 M urea, 1%-10% mannitol, 1-5 M NaCl, pH 8.5-8.9. 
     
     
         9 . The polypeptide according to  claim 6 , wherein the equilibration buffer B comprises 10-50 mM phosphate buffer PB, 1-5M urea, 1%-10% mannitol, pH 6.0-6.5. 
     
     
         10 . The polypeptide according to  claim 6 , wherein the elution buffer B comprises 10-50 mM phosphate buffer PB, 1-5M urea, 1%-10% mannitol, 1-5 M NaCl, pH 6.0-6.5. 
     
     
         11 . The polypeptide according to  claim 6 , wherein the sequence is subjected to galactosylated modification. 
     
     
         12 . The polypeptide according to  claim 11 , wherein the galactosylated modification is modified by artificial modification in vitro or by in vivo expression in a eukaryotic organism. 
     
     
         13 . The polypeptide according to  claim 12 , wherein the artificial modification is modified with PEG. 
     
     
         14 . A method for producing recombinant human bone morphogenetic protein-2, comprising the following steps:
 1) designing DNA sequence encoding recombinant human bone morphogenetic protein-2;   2) constructing an expression vector to transform  E. coli  host cells;   3) screening positive clones for culture and inducing the expression of the target protein;   4) renaturing and purifying the expression product to obtain the recombinant human bone morphogenetic protein-2,   wherein the DNA sequence is shown in SEQ ID NO: 3.   
     
     
         15 . The method according to  claim 14 , wherein a dilution method is used for renaturation in the step (4) and the final concentration of the protein in the renaturation buffer is controlled at 0.05-0.5 mg/ml. 
     
     
         16 . The method according to  claim 14 , wherein multi-step ion exchange chromatography is used for purification in step (4) and the steps are as follows:
 1) loading the renatured recombinant human bone morphogenetic protein-2 solution onto a well-balanced strong anion column, and rinsing with equilibration buffer A to reach the baseline after loading;   2) performing stepwise salt-gradient elution using the elution buffer A and collecting the main peak;   3) mixing the target peak solution collected from the strong anion column and loading onto a weak cation column, and rinsing with equilibration buffer B to reach the baseline after loading;   4) performing stepwise salt-gradient elution using the elution buffer B and collecting the main peak.   
     
     
         17 . The method according to  claim 16 , wherein the equilibration buffer A comprises 10-50 mM Tris-HCl, 1-5 M urea, 1%-10% mannitol, pH 8.5-8.9; the elution buffer A comprises 10-50 mM Tris-HCl, 1-5 M urea, 1%-10% mannitol, 1-5 M NaCl, pH 8.5-8.9; the equilibration buffer B comprises 10-50 mM phosphate buffer PB, 1-5M urea, 1%-10% mannitol, pH 6.0-6.5; and the elution buffer B comprises 10-50 mM phosphate buffer PB, 1-5M urea, 1%-10% mannitol, 1-5 M NaCl, pH 6.0-6.5. 
     
     
         18 . The method according to  claim 14 , wherein the sequence is subjected to galactosylated modification. 
     
     
         19 . The method according to  claim 18 , wherein the galactosylated modification is modified by artificial modification in vitro or by in vivo expression in a eukaryotic organism. 
     
     
         20 . The method according to  claim 19 , wherein the artificial modification is modified with PEG.