U.S. patent application number 16/518378 was filed with the patent office on 2020-01-30 for polypeptide of recombinant human bone morphogenetic protein-2.
The applicant listed for this patent is Zhejiang Rising Biotechnology Co., Ltd. Invention is credited to Jianxin CAO.
Application Number | 20200031896 16/518378 |
Document ID | / |
Family ID | 69178978 |
Filed Date | 2020-01-30 |
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United States Patent
Application |
20200031896 |
Kind Code |
A1 |
CAO; Jianxin |
January 30, 2020 |
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.
Inventors: |
CAO; Jianxin; (Hangzhou,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zhejiang Rising Biotechnology Co., Ltd |
Hangzhou |
|
CN |
|
|
Family ID: |
69178978 |
Appl. No.: |
16/518378 |
Filed: |
July 22, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 1/18 20130101; C07K
14/51 20130101 |
International
Class: |
C07K 14/51 20060101
C07K014/51; C07K 1/18 20060101 C07K001/18 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 24, 2018 |
CN |
201810818738.7 |
Claims
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.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application claims the benefit of Chinese Patent
Application No. 201810818738.7, filed on Jul. 24, 2018. The content
of the application including all tables, diagrams and claims is
incorporated hereby as reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a field of biotechnology,
and in particular to 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.
BACKGROUND OF THE INVENTION
[0003] Bone morphogenetic protein (BMP) is a member of the
transforming growth factor (TGF)-.beta. superfamily. With unique
osteoinductive activity, BMP has important functions in the repair
of bone segment defects of bones and joints and spinal fusion, etc.
At present, a variety of BMPs have been isolated from bone matrix
of various animals such as cattle, pigs, sheep, rabbits, mice and
human, etc., of which, human bone morphogenetic protein-2 (BMP-2)
has the best induced osteogenic effect and it is an indispensable
factor in the formation of the skeletal system. BMP-2 is an acidic
glycoprotein containing 396 amino acid residues consisting of a
N-terminal signal peptide, an intermediate propeptide and a
C-terminal mature peptide. The mature peptide consists of 114 amino
acids and is a part that exerts a function of osteogenic induction,
which contains seven cysteine residues, of which 6 cysteine
residues form an intramolecular disulfide bond and the other is
used to form an intermolecular disulfide bond. The homodimer or
heterodimer of the mature peptide is biologically active, wherein
monomers form three pairs of disulfide bonds, forming a pair of
interchain disulfide bonds between two monomers. The correct
pairing of disulfide bonds has a direct impact on the biological
activity and stability. In addition, a glycosylation site exists on
the asparagine residue at position 55 of the mature peptide, but
its biological activity is not affected by glycosylation. BMP-2 has
been approved by the US FDA for bone injury, frontal repair, and
spinal fusion. However, the source of fresh human bones is limited
and bone BMP-2 level is low in the bone, only 1-2 .mu.g per kg of
cortical bone, which is difficult to meet clinical needs. Moreover,
its extraction process is complicated, and its purity and activity
could not be guaranteed. Therefore, it has become a research
hotspot to produce this protein by genetic recombinant expression
using the genetic engineering techniques.
[0004] Currently, rhBMP-2 is prepared mainly through eukaryotic
expression vector and prokaryotic expression vector. Eukaryotic
expression systems are mainly used at abroad, such as CHO, COS and
baculovirus systems. Most eukaryotic expression products are
secretory type and undergo glycosylation processing; rhBMP-2 is a
secretory protein and has glycosylation sites, so the eukaryotic
expression system is an ideal expression system. The product
prepared by a eukaryotic expression system has high activity, but
it has the drawbacks of low expression level, high production cost,
and unsuitable for mass production, etc. At present, medical
rhBMP-2 at home and abroad is mainly produced by a prokaryotic
expression system with Escherichia coli as a host, to obtain
fragments with different lengths of rhBMP-2 mature peptides. As
prokaryotic expression systems do not have the process of
post-translational modification of polypeptide chains by eukaryotic
cells, the eukaryotic gene proteins cannot be correctly folded and
post-translationally processed (such as forming disulfide bonds,
glycosylation, polymerization, protease degradation, etc.), which
restricts their biological activity and availability. In addition,
eukaryotic proteins expressed in prokaryotic expression systems
usually aggregate into insoluble inclusion bodies in the cytoplasm
of bacteria in an inactive manner, which must undergo denaturation,
renaturation to restore their natural activities. Chinese Patent
CN1215171C discloses a method for producing a truncated recombinant
human bone morphogenetic protein-2 mature peptide, which is
asynthetically encoded BMP-2 nucleotide sequence with 107 amino
acid residues in C-terminal, an initiation codon ATG is added
before the first amino acid codon and the second amino acid
arginine (R) is mutated to a lysine (K) residue, with amino acid
sequence of MKKLKSSCKR HPLYVDFSDV GWNDWIVAPP GYHAFYCHGE CPFPLADHLN
STNHAIVQTL VNSVNSKIPK ACCVPTELSA ISMLYLDENE KVVLKNYQDM VVEGCGCR
(SEQ ID NO:6). The amount of human truncated BMP-2 produced by
genetically engineered bacteria is 30-50% of the total soluble
protein of the bacteria, with osteogenic activity. Chinese
invention patent CN101787369B discloses an optimized recombinant
human bone morphogenetic protein-2 DNA sequence and the preparation
and use of encoded protein thereof. In the invention, by optimizing
DNA sequence, rhBMP-2 with complete mature peptide (114 amino acid
residues) is obtained, and the product purity exceeds 95%, after
purification, the yield of dimer is about 21%. The prepared rhBMP-2
using the existing several kinds of rhBMP-2 mature peptide
fragments has poor renaturation and purification effects, and the
final product has low biological activity. To solve the above
problems, it is required to analyze the mature peptide gene using
genetic recombination technology, optimize the nucleotide sequence
of the target gene without affecting and changing the molecular
structure of proteins, and screen out novel amino acid sequence of
rhBMP-2 that can express good ectopic induced osteogenic activity
and has good renaturation and purification effect.
SUMMARY OF THE INVENTION
[0005] An object of the present invention is to provide a novel
amino acid sequence of recombinant human bone morphogenetic
protein-2 (rhBMP-2) such that the expressed protein has good
renaturation and purification effect and has high biological
activity and bone repair ability.
[0006] In order to solve the above technical problems, the present
invention adopts the following technical solutions.
[0007] The amino acid sequence of rhBMP-2 is optimized and the
novel amino acid sequence of recombinant human bone morphogenetic
protein-2 is as follows:
TABLE-US-00001 (SEQ ID NO: 1) KRLK SSCKR HPLYV DFSDV GWNDW IVAPP
GYHAF YCHGE CPFPL ADHLN STNHA IVQTL VNSVN SKIPK ACCVP TELSA ISMLY
LDENE KVVLK NYQDMVVEGC GCR.
[0008] Further, the N-terminal of the above protein sequence is
added with methionine M encoded by the initiation codon (AUG). The
amino acid sequence is as follows:
TABLE-US-00002 (SEQ ID NO: 2) MKRLK SSCKR HPLYV DFSDV GWNDW IVAPP
GYHAF YCHGE CPFPL ADHLN STNHA IVQTL VNSVN SKIPK ACCVP TELSA ISMLY
LDENE KVVLK NYQDM VVEGC GCR.
[0009] The amino acid sequence also includes that modified by
glycosylation, for example, amino acid sequences modified by
PEG.
[0010] By optimizing the codons, all rare codons are removed to
become E. coli-preferred codons. The optimized DNA sequences are as
follows:
TABLE-US-00003 (SEQ ID NO: 3)
ATGAAACGTCTGAAAAGCAGCTGCAAACGTCACCCGCTGTACGTTGATTT
CAGCGATGTTGGCTGGAACGATTGGATCGTTGCGCCGCCGGGCTACCACG
CGTTCTACTGCCACGGCGAATGCCCGTTCCCGCTGGCGGATCACCTGAAC
AGCACCAACCACGCGATCGTTCAGACCCTGGTTAACAGCGTTAACAGCAA
AATCCCGAAAGCGTGCTGCGTTCCGACCGAACTGTCTGCGATCTCAATGC
TGTACCTGGATGAAAACGAAAAAGTTGTTCTGAAAAACTACCAGGATATG
GTTGTTGAAGGTTGCGGTTGCCGTTAA.
[0011] Another object of the present invention is to provide a
method for producing recombinant human bone morphogenetic
protein-2, comprising the following steps: (1) designing an amino
acid sequence and a 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.
[0012] Preferably, the vector is expressed in eukaryotic cells, for
example CHO cells. The expressions of protein modified by
glycosylation in eukaryotic cells and the amino acid sequence with
activity after modified by glycosylation are also within the scope
of the present invention. Alternatively, the protein is
artificially modified in vitro, for example, modified with PEG, to
greatly increase its stability. Our experiments have found that the
stability of the modified proteins that are expressed in vitro is
increased by about 35-50% after artificial modification.
[0013] Preferably, a dilution method is used for renaturation and
the final concentration of the protein in the renaturation buffer
is controlled at 0.05-0.5 mg/ml.
[0014] Preferably, in step (4), multi-step ion exchange
chromatography is used for purification, 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.
[0015] Preferably, 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.
[0016] In the present invention, by optimizing the nucleotide
sequence of rhBMP-2 gene to screen out a novel amino acid sequence
capable of expressing rhBMP-2 with good ectopic osteoinductive
activity and good renaturation and purification effect. The
engineered bacteria constructed by the 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. The yield of purified
target protein is greater than 60% or about 60% and the purity is
higher than 98%. The expressed recombinant rhBMP-2 has higher
osteoinductive activity, presenting broad market prospect.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a profile of pET28 plasmid used in the present
invention.
[0018] FIG. 2 is a multiple cloning site of plasmid pET28.
[0019] FIG. 3 is a SDS-PAGE electrophoresis pattern of rhBMP-2
induced expression.
[0020] FIG. 4 is a renaturation result of SDS-PAGE electrophoresis
analysis, wherein 1: molecular weight standard; 2-7: renatured
rhBMP-2 dimer.
[0021] FIGS. 5A and 5B show a purity result of SDS-PAGE
electrophoresis analysis, wherein FIG. 5A is a target peak
electrophoresis pattern in strong anion column, FIG. 5B is a target
peak electrophoresis pattern in weak cation column, 1: molecular
weight standard; 2-10: collected target peaks.
[0022] FIG. 6 is a capillary electrophoresis purity analysis
pattern of rhBMP-2, detected at UV214 nm.
[0023] FIGS. 7A and 7B show mouse muscle ectopic osteogenesis
radiology examination of rhBMP-2 and induced new bones, wherein, 1
is an induced osteogenic effect chart of 108-peptide recombinant
rhBMP-2, 2 is an induced osteogenic effect chart of rhBMP-2
expressed by amino acid sequences in the present invention, 3 is an
induced osteogenic effect chart of 109-peptide recombinant rhBMP-2,
4 is an induced osteogenic effect chart of 115-peptide recombinant
rhBMP-2.
DETAILED DESCRIPTION
[0024] The present invention is further described in conjunction
with particular embodiments, which are merely illustrative of the
invention, but are not intended to limit the invention. Unless
otherwise indicated, the terms used herein have the same meaning as
commonly understood by those skilled in the art.
[0025] In the present invention, all percentages are expressed in
weight/weight (w/w) unless otherwise specified. All equipment and
materials are commercially available or commonly used in the
industry. Unless otherwise specified, the method used in the
embodiments is a general technology in the art. Since the codon
(ATG) of methionine M is the initiation codon of protein
translation process, the amino acid sequence SEQ ID NO: 2
containing the methionine M at the N-terminal is used for
experiments.
Example 1 Design of Novel Amino Acid Sequence of rhBMP-2 and
Construction of Recombinant rhBMP-2 Expression Vector
[0026] 1. In One Embodiment, the Polypeptide of rhBMP-2 has the
Amino Acid Sequence as Follows:
TABLE-US-00004 (SEQ ID NO: 1) KRLK SSCKR HPLYV DFSDV GWNDW IVAPP
GYHAF YCHGE CPFPL ADHLN STNHA IVQTL VNSVN SKIPK ACCVP TELSA ISMLY
LDENE KVVLK NYQDM VVEGC GCR.
[0027] Further, the N-terminal of the above amino acid sequence is
added with methionine M encoded by the initiation codon (ATG). The
amino acid sequence is as follows:
TABLE-US-00005 (SEQ ID NO: 2) MKRLK SSCKR HPLYV DFSDV GWNDW IVAPP
GYHAF YCHGE CPFPL ADHLN STNHA IVQTL VNSVN SKIPK ACCVP TELSA ISMLY
LDENE KVVLK NYQDM VVEGC GCR.
[0028] Further, by optimizing the codons, all rare codons are
removed to become E. coli-preferred codons, at the same time, the
restriction site BamHI (GGATCC) is added at the terminal. The
optimized DNA sequences are as follows:
TABLE-US-00006 (SEQ ID NO: 3)
ATGAAACGTCTGAAAAGCAGCTGCAAACGTCACCCGCTGTACGTTGATTT
CAGCGATGTTGGCTGGAACGATTGGATCGTTGCGCCGCCGGGCTACCACG
CGTTCTACTGCCACGGCGAATGCCCGTTCCCGCTGGCGGATCACCTGAAC
AGCACCAACCACGCGATCGTTCAGACCCTGGTTAACAGCGTTAACAGCAA
AATCCCGAAAGCGTGCTGCGTTCCGACCGAACTGTCTGCGATCTCAATGC
TGTACCTGGATGAAAACGAAAAAGTTGTTCTGAAAAACTACCAGGATATG
GTTGTTGAAGGTTGCGGTTGCCGTTAA.
[0029] 2. Construction of Expression Vector, Transformation of Host
Cells of Escherichia coli, Construction of Expression Engineering
Bacteria and Process of Recombinant Protein Expression
[0030] The prokaryotic expression vector pET28a was selected as the
expression vector of recombinant rhBMP-2 protein, and the plasmid
map of pET28a was shown in FIG. 1. The gene fragments described in
SEQ ID NO: 3 were inserted into an expression vector after
digestion to complete the construction of vector. The pET28a vector
had a T7lac promoter, T7 could ensure efficient expression of the
exogenous inserted gene, and the lac lactose operon enabled the
gene to maintain a low background expression in the absence of an
inductive agent. The multiple cloning site of the pET28a vector was
shown in FIG. 2.
[0031] (1) Construction of an Expression Vector
[0032] The pET28a vector was digested with NcoI and filled in, and
then digested with BamHI. At the same time, the synthesized rhBMP-2
(SEQ ID NO:3) was digested with BamHI. The reaction system: total
reaction volume 20 .mu.L, ddw 12 .mu.L, plasmid DNA 5 .mu.L,
10.times. Buffer K2 .mu.L, BamHI 1 .mu.L.
[0033] The digested plasmid was separated on 1% agarose gel, and
the target fragments were cleaved from the gel, and target
fragments were recovered using Axygen's gel recovery kit.
[0034] After recovering DNA fragments, the pET28a vector fragment
and the rhBMP-2 gene fragment were ligated with T4 DNA ligase. The
ligation reaction system was: total reaction volume 10 .mu.L,
pET28a 4 .mu.L, rhBMP-2 4 .mu.L, 10.times.DNA ligase Buffer 1
.mu.L, T4 DNA ligase 1 .mu.L. The ligation reaction was carried out
at 16.degree. C. for 1 hour.
[0035] (2) Transformation of Escherichia coli Host Cells
[0036] After the ligation reaction was completed, the recombinant
plasmid was transformed into E. coli DH5a cells by heat shock
transformation. The transformation process was as follows: the
ligation product was added to E. coli competent cells, placed on
ice for 30 min, then the competent cells were placed in a
42.degree. C. water bath for heat shock for 90 sec, and then a
centrifuge tube was quickly inserted into the ice, after 1-2 min, 1
mL of LB medium was added, and the centrifuge tube was placed in a
shaker and incubated for 1 hour at 37.degree. C. and 160 rpm.
Escherichia coli was collected by centrifugation and coated to a
kanamycin-containing LB solid medium, and a plate was placed in a
37.degree. C. biochemical incubator for culture overnight. On the
next day, a single clone was picked and PCR was used to identify
whether a foreign gene was inserted. The PCR reaction system was:
total reaction volume 20 .mu.L, ddw 9 .mu.L, 2.times.Taq pre-mix 10
.mu.L, T7 promoter (10 .mu.M) 0.5 .mu.L, rhBMP-2 reverse primer (10
.mu.M) 0.5 .mu.L.
[0037] The cells of partially picked monoclonal colonies were taken
and added to the PCR reaction system for PCR reaction on a Bio-Rad
PCR instrument. The reaction procedure was as follows:
pre-denaturation at 94.degree. C. for 5 min; denaturation at
94.degree. C. for 30 sec, annealing at 55.degree. C. for 30 sec,
extension at 72.degree. C. for 30 sec, after 35 cycles, extension
at 72.degree. C. for 15 min.
[0038] The correct monoclone identified by PCR was sequenced with
T7promoter primers to detect the correctness of the rhBMP-2
sequence. The correctly sequenced monoclone was re-inoculated into
a kanamycin-containing LB medium, and cultured in a shaker at
37.degree. C., 250 rpm, and then the plasmid was extracted for use.
The recombinant plasmid was named pET28a-rhBMP-2.
[0039] (3) Transformation of rhBMP-2 Expression Vector into E. coli
Expression Strain
[0040] The recombinant rhBMP-2 protein was expressed by BL21 (DE3)
expression strain. The recombinant plasmid pET28a-rhBMP-2 was added
to E. coli BL21 (DE3) competent cells, placed on ice for 30
minutes, and the water bath was heated to 42.degree. C. for use.
After placed on ice for 30 min, the centrifuge tube containing
Escherichia coli BL21 (DE3) was placed in a 42.degree. C. water
bath for heat shock for 90 sec, and then a centrifuge tube was
quickly inserted into the ice, after 1-2 min, 1 mL of LB medium was
added, and the centrifuge tube was placed in a shaker for culture
for 1 hour at 37.degree. C. and 160 rpm. Escherichia coli BL21
(DE3) was collected by centrifugation and coated to a
kanamycin-containing LB solid medium, and a plate was placed in a
37.degree. C. biochemical incubator for culture overnight. On the
next day, a single clone was picked and PCR was used to identify
whether BL21(DE3) was transferred to the recombinant plasmid. The
PCR reaction system was: total reaction volume 20 .mu.L, ddw 9
.mu.L, 2.times.Taq pre-mix 10 .mu.L, T7 promoter (10 .mu.M) 0.5
.mu.L, rhBMP-2 reverse primer (10 .mu.M) 0.5 .mu.L.
[0041] The cells of partially picked monoclonal colonies were taken
and added to the PCR reaction system for PCR reaction on a Bio-Rad
PCR instrument. The reaction procedure was as follows:
pre-denaturation at 94.degree. C. for 5 min; denaturation at
94.degree. C. for 30 sec, annealing at 55.degree. C. for 30 sec,
extension at 72.degree. C. for 30 sec, after 35 cycles, extension
at 72.degree. C. for 15 min.
[0042] The correct monoclone identified by PCR was transferred to a
kanamycin-containing LB medium, and cultured in a shaker at
37.degree. C. and 250 rpm. When the OD600 of the bacteria solution
reaches 0.6-0.8, the bacteria solution was taken out and 80%
sterile glycerol was added to a final concentration of 10%, and
then stored at -20.degree. C. for later use.
[0043] (4) Expression of Recombinant rhBMP-2
[0044] The BL21 (DE3) strain containing pET28a-rhBMP-2 stored in
glycerin was inoculated into a kanamycin-containing LB liquid
medium at a volume ratio of 1:1000, and shake-flask cultured to the
logarithmic phase at the condition of 37.degree. C. and 100-200
rpm. The induced expression of rhBMP-2 protein was performed by
different concentrations of inductive agent IPTG at different
induction times and different induction temperatures. After the
induction culture, the solution was centrifuged for 20 min at 3500
rpm and 4.degree. C. to collect the thallus, and then the protein
expression level was analyzed by SDS-PAGE. The combination with
highest expression of rhBMP-2 was selected as the experimental
parameters with the optimized induction expression of rhBMP-2
protein. The experiments showed that, the protein expression level
was highest (about 55%) when the IPTG concentration was 1.0 mM, the
induction temperature was 37.degree. C. and the induction time was
5 h (FIG. 3).
Example 2 Renaturation and Purification of Recombinant rhBMP-2
[0045] (1) Renaturation
[0046] After the bacteria were disrupted by sonication, the
inclusion bodies formed by the rhBMP-2 protein were extracted, then
the inclusion bodies were washed, and dissolved with a denaturant
guanidine hydrochloride. The renaturation was carried out by the
dilution method, and the final concentration of the renaturation
liquid protein was controlled at 0.05-0.5 mg/ml. The rhBMP-2
denatured protein solution was continuously and slowly added to the
renaturation butter for dilution and renaturation. After addition,
the mixture was slowly stirred for a few minutes, and allowed to
stand for 2-20 days for renaturation. After renaturation, the
rhBMP-2 mature peptide monomer forms a dimer, and all monomers form
three pairs of intramolecular disulfide bonds, and two monomers
form a dimer through an intermolecular disulfide bond. After
renaturation, samples were taken for SDS-PAGE electrophoresis, to
judge the renaturation rate and renaturation effect.
[0047] According to the results of FIG. 4, 150.2 mg of the
inclusion body protein was obtained by 500 mL of induced bacterial
solution, and the yield of target protein rhBMP was high (about
80%).
[0048] (2) Purification
[0049] The multi-step ion exchange chromatography was used for
purification and the specific purification steps were as
follows:
[0050] 1) The renatured rhBMP-2 solution was loaded onto a
well-balanced strong anion column, and then rinsed with
equilibration buffer A to reach the baseline after loading. The
equilibration buffer A comprised 10-50 mM Tris-HCl, 1-5 M urea,
1%-10% mannitol, pH 8.5-8.9.
[0051] 2) A stepwise salt-gradient elution was performed using the
elution buffer A and the main peak was collected for SDS-PAGE
analysis (FIG. 5A). The elution buffer A comprised 10-50 mM
Tris-HCl, 1-5 M urea, 1%-10% mannitol, 1-5 M NaCl, pH 8.5-8.9.
[0052] 3) The target peak solution collected from the strong anion
column was mixed and loaded onto a weak cation column, and then
rinsed with equilibration buffer B to reach the baseline after
loading. The equilibration buffer B comprised 10-50 mM phosphate
buffer PB, 1-5M urea, 1%-10% mannitol, pH 6.0-6.5.
[0053] 4) A stepwise salt-gradient elution was performed using the
elution buffer B and the main peak was collected for SDS-PAGE
analysis. The elution buffer B comprised 10-50 mM phosphate buffer
PB, 1-5M urea, 1%-10% mannitol, 1-5 M NaCl, pH 6.0-6.5.
[0054] FIG. 5B showed that the target protein recovered was a
single band with purity higher than 98%. Data analysis showed that
the total soluble protein of the renaturation solution was 150.2
mg, and the yield of purified rhBMP-2 was 89.3 mg, with a yield
rate of about 60%.
[0055] The capillary electrophoresis analysis showed that the
sample purity was 98.5% (FIG. 6).
Example 3 In Vitro Activity Assay of Recombinant rhBMP-2 in C2C12
Cells
[0056] C2C12 cells (mesenchymal stem cells) were cultured in DMEM
high glucose medium (containing 10% fetal bovine serum, 100 U/mL
penicillin and 100 g/mL streptomycin) under the conditions of
37.degree. C. and 5% CO2 and digested with 0.25% trypsin for
passage every 1-2d. C2C12 cells were inoculated in a 24-well plate,
and when cell fusion reached around 30%, recombinant rhBMP-2 was
added for continuous culture. After 5 days of culture, the medium
was discarded and washed twice with pre-cooled PBS buffer. After
lysed with lysate for 5 min, the supernatant was centrifuged and
the activity of alkaline phosphatase (ALP) was quantitatively
determined according to the instructions of the kit.
[0057] Controlled Trial:
[0058] In order to compare the biological activities of recombinant
rhBMP-2 having the novel amino acid sequences of the present
invention, three kinds of reported polypeptides were used for
controlled trial. The engineered bacteria were constructed
according to the reported amino acid sequences and gene sequences
and the corresponding recombinant rhBMP-2 was obtained by induced
expression, renaturation and purification using the method
described herein. The amino acid sequences of the three
polypeptides selected were as follows.
[0059] 1. 115-peptide (containing methionine in the N terminal,
disclosed in the invention patent CN101787369B)
TABLE-US-00007 (SEQ ID NO: 4) MQAKHKQRKR LKSSCKRHPL YVDFSDVGWN
DWIVAPPGYH AFYCHGECPF PLADHLNSTNHAIVQTLVNS VNSKIPKACC VPTELSAISM
LYLDENEKVV LKNYQDMVVE GCGCR
[0060] 2. 109-peptide (containing methinonine in the N
terminal)
TABLE-US-00008 (SEQ ID NO: 5) MRKRLKSSCK RHPLYVDFSD VGWNDWIVAP
PGYHAFYCHG ECPFPLADHL NSTNHAIVQTLVNSVNSKIP KACCVPTELS AISMLYLDEN
EKVVLKNYQD MVVEGCGCR
[0061] 3. 108-peptide (containing methionine in the N terminal,
disclosed in the invention patent CN1215171C)
TABLE-US-00009 (SEQ ID NO: 6) MKKLKSSCKR HPLYVDFSDV GWNDWIVAPP
GYHAFYCHGE CPFPLADHLN STNHAIVQTLVNSVNSKIPK ACCVPTELSA ISMLYLDENE
KVVLKNYQDM VVEGCGCR
[0062] 4. The sequences designed in the present invention
(containing methionine in the N terminal)
TABLE-US-00010 (SEQ ID NO: 2) MKRLK SSCKR HPLYV DFSDV GWNDW IVAPP
GYHAF YCHGE CPFPL ADHLN STNHA IVQTL VNSVN SKIPK ACCVP TELSA ISMLY
LDENE KVVLK NYQDM VVEGC GCR.
[0063] Compared with the 115-peptide, the amino acid sequence of
the present invention was a 107 amino acid sequences truncated from
amino acid residues of the rhBMP-2 mature peptide; 109-peptide was
108 amino acid sequences truncated from amino acid residues of the
rhBMP-2 mature peptide; and 108-peptide was a 107 amino acid
sequences truncated from amino acid residues of the rhBMP-2 mature
peptide and the second amino acid arginine residue (R) was mutated
to a lysine residue (K).
[0064] The test results were shown in the table below.
TABLE-US-00011 TABLE 1 Activity assay in C2C12 cells Experiment
group ALP activity 115-peptide 43% 109-peptide 45% 108-peptide 78%
The present invention 98%
[0065] The above results showed that the activity of recombinant
rhBMP-2 comprising the amino acid sequence of the present invention
was much higher than that of the other three kinds of peptide in
the C2C12 cells, indicating that the C2C12 cells could be better
differentiated to osteoblasts under the action of recombinant
rhBMP-2 designed in the present invention.
[0066] Some researchers believe that the closer the length of
rhBMP-2 to a complete mature peptide, the better the osteogenic
activity (Lin Song et al., Acta Biochimica et Biophysica Sinica,
1996, 28(1): 8). In the present invention, it was surprisingly
found that the truncated polypeptide of the present invention had
much higher activity to promote differentiation of C2C12 cells than
mature peptides, possibly because the peptide chain of the mature
peptide was longer and the peptide chain was liable to form a
helical structure under the action of hydrogen bonds so that the
active site on the peptide chain was easily embedded in the
hydrophobic cavity, affecting its binding to the receptor on the
cell membrane surface, and the shortened peptide chain would reduce
the effect of steric hindrance; another reason may be that the
nitrogen-terminal peptide segment of the mature peptide had no
activity, or the nitrogen-terminal peptide segment of the mature
peptide had endoprotease activity, which would cause degradation of
rhBMP-2 and affect its activity. The specific reasons need to be
further identified.
[0067] The 109-peptide was 108 amino acid sequences truncated from
amino acid residues of the rhBMP-2 mature peptide. Compared with
the present invention, its peptide chain nitrogen terminal
contained one more arginine residue (R). Experiments have found
that its activity to promote the differentiation of C2C12 cells is
significantly lower than that of the present invention, possibly
because the presence of the arginine residue at position 108 of the
mature peptide would cause the degradation of rhBMP-2. Further,
compared with the present invention, the second amino acid arginine
residue (R) of peptide chain of the disclosed 108-peptide was
mutated to a lysine residue (K), which would reduce the activity of
the recombinant protein, thus, it could be inferred that the
mutation of the arginine residue at position 106 of the mature
peptide of rhBMP-2 would affect its biological activity.
Example 4 Effect of Three Vectors on Mouse Muscle Ectopic
Osteogenesis Induced by Recombinant RhBMP-2
[0068] The recombinant rhBMP-2 aqueous solution was mixed with
high-concentration gelatin, chitosan or natural coral, and the
recombinant rhBMP-2 was uniformly combined with the three vectors
to form a composite material by vacuumizing. Each vector contained
100 .mu.g rhBMP-2, which was lyophilized and disinfected by
ethylene oxide for later use.
[0069] Six normal ICR male mice were randomly divided into three
groups, two mice in each group. After anesthesia with 1% sodium
pentobarbital, the hind limbs were depilated and disinfected, and
the skin was cut to separate the spatium intermusculare, and then
implanted with a certain amount of the above composite material.
Among them, the implanted materials in the first group used high
concentration gelatin as a vector, and chitosan was used as a
vector in the second group, and natural coral was used as a vector
in the third group. Mice were given antibiotics to prevent
infection, and then muscles and skin were sutured layer by layer,
and the wounds were disinfected for normal feeding. Three weeks
after the implantation experiment, the mice underwent radiological
examination (X-ray) to observe if bone tissues appeared in the hind
limbs of the mice. The mice were sacrificed, bone tissues of the
implanted area were taken out, and the bones contained were
weighed.
[0070] The radiological examination showed that the ectopic
osteogenesis of the first group of mice was obvious with tight
adhesion to autologous bone of mice. Further bone weighing found
that, the average bone weight was 0.706 g when a high concentration
of gelatin was used as the vector; the average bone weight was
0.266 g when chitosan was used as the vector; and the average bone
weight was 0.345 g when the natural coral was used as the vector.
The above results showed that, a high-concentration of gelatin
could promote ectopic osteogenesis, possibly because the molecular
structure of gelatin was more suitable for osteogenesis, so that
rhBMP-2 was in uniform contact with adjacent tissues, to
continuously maintain its activity and prolong the reaction
time.
Example 5 Mouse Muscle Ectopic Osteogenesis Induced by Four Kinds
of Recombinant rhBMP-2
[0071] In order to compare the biological activity of recombinant
rhBMP-2 of the novel amino acid sequence of the present invention,
the inventors conducted controlled trial using the recombinant
rhBMP-2 of three kinds of reported polypeptides described in
Example 3 (the recombinant protein was prepared according to the
method described in the present invention). In the experiments, a
high-concentration gelatin was used as a vector to form a composite
material with four kinds of rhBMP-2. Each kind of composite
material contained 100 .mu.g of corresponding rhBMP-2.
[0072] Eight normal ICR male mice were randomly divided into four
groups, two mice in each group. After anesthesia with 1% sodium
pentobarbital, the hind limbs were depilated and disinfected, and
the skin was cut to separate the spatium intermusculare, then
implanted with a certain amount of the above four kinds of
composite material. Three weeks later, radiological examination
(X-ray) and osteogenesis testing were performed.
[0073] The radiological examination results were shown in FIG. 7A.
The mice in the second group had obvious ectopic osteogenesis, with
compact adhesion to autologous bones of mice. Further bone weighing
found that, the rhBMP-2 prepared by in the present invention had
better osteogenic induction activity than the other three
recombinant proteins.
TABLE-US-00012 TABLE 2 Mouse muscle ectopic osteogenesis induced by
four kinds of rhBMP-2 Amino acid sequence of Average osteogenesis
Experiment Group rhBMP-2 weight/g 1 108-peptide 0.385 2 The present
invention 0.580 3 109-peptide 0.240 4 115-peptide 0.185
[0074] The rhBMP promotes new bone formation by inducing
differentiation of undifferentiated mesenchymal cell C2C12 to form
osteoblasts. The recombinant rhBMP-2 of the amino acid sequence of
the present invention has a significant activity to promote the
differentiation of C2C12 cells compared with the other three
recombinant rhBMPs, exhibiting remarkable osteogenic activity.
[0075] The above technical solutions are merely illustrative of the
preferred embodiments of the present invention, and are not to be
construed as limiting the scope of the present invention. All
modifications and improvements made based on the present invention
are within the scope of protection of the present invention.
[0076] The contents of articles, patents, patent applications, and
all other documents and electronically available information
described or recited herein are hereby incorporated by reference in
their entirety as if individually pointed out for reference for
each publication. The applicant reserves the right to incorporate
any and all materials and information from any such article,
patent, patent application or other document into this application.
Sequence CWU 1
1
61107PRTArtificial Sequencesynthetic 1Lys Arg Leu Lys Ser Ser Cys
Lys Arg His Pro Leu Tyr Val Asp Phe1 5 10 15Ser Asp Val Gly Trp Asn
Asp Trp Ile Val Ala Pro Pro Gly Tyr His 20 25 30Ala Phe Tyr Cys His
Gly Glu Cys Pro Phe Pro Leu Ala Asp His Leu 35 40 45Asn Ser Thr Asn
His Ala Ile Val Gln Thr Leu Val Asn Ser Val Asn 50 55 60Ser Lys Ile
Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile65 70 75 80Ser
Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr 85 90
95Gln Asp Met Val Val Glu Gly Cys Gly Cys Arg 100
1052108PRTArtificial Sequencesynthetic 2Met Lys Arg Leu Lys Ser Ser
Cys Lys Arg His Pro Leu Tyr Val Asp1 5 10 15Phe Ser Asp Val Gly Trp
Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr 20 25 30His Ala Phe Tyr Cys
His Gly Glu Cys Pro Phe Pro Leu Ala Asp His 35 40 45Leu Asn Ser Thr
Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val 50 55 60Asn Ser Lys
Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala65 70 75 80Ile
Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn 85 90
95Tyr Gln Asp Met Val Val Glu Gly Cys Gly Cys Arg 100
1053327DNAArtificial Sequencesynthetic 3atgaaacgtc tgaaaagcag
ctgcaaacgt cacccgctgt acgttgattt cagcgatgtt 60ggctggaacg attggatcgt
tgcgccgccg ggctaccacg cgttctactg ccacggcgaa 120tgcccgttcc
cgctggcgga tcacctgaac agcaccaacc acgcgatcgt tcagaccctg
180gttaacagcg ttaacagcaa aatcccgaaa gcgtgctgcg ttccgaccga
actgtctgcg 240atctcaatgc tgtacctgga tgaaaacgaa aaagttgttc
tgaaaaacta ccaggatatg 300gttgttgaag gttgcggttg ccgttaa
3274115PRTArtificial Sequencesynthetic 4Met Gln Ala Lys His Lys Gln
Arg Lys Arg Leu Lys Ser Ser Cys Lys1 5 10 15Arg His Pro Leu Tyr Val
Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30Ile Val Ala Pro Pro
Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys 35 40 45Pro Phe Pro Leu
Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val 50 55 60Gln Thr Leu
Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys65 70 75 80Val
Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn 85 90
95Glu Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu Gly Cys
100 105 110Gly Cys Arg 1155109PRTArtificial Sequencesynthetic 5Met
Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg His Pro Leu Tyr Val1 5 10
15Asp Phe Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gly
20 25 30Tyr His Ala Phe Tyr Cys His Gly Glu Cys Pro Phe Pro Leu Ala
Asp 35 40 45His Leu Asn Ser Thr Asn His Ala Ile Val Gln Thr Leu Val
Asn Ser 50 55 60Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr
Glu Leu Ser65 70 75 80Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu
Lys Val Val Leu Lys 85 90 95Asn Tyr Gln Asp Met Val Val Glu Gly Cys
Gly Cys Arg 100 1056108PRTArtificial Sequencesynthetic 6Met Lys Lys
Leu Lys Ser Ser Cys Lys Arg His Pro Leu Tyr Val Asp1 5 10 15Phe Ser
Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr 20 25 30His
Ala Phe Tyr Cys His Gly Glu Cys Pro Phe Pro Leu Ala Asp His 35 40
45Leu Asn Ser Thr Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val
50 55 60Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser
Ala65 70 75 80Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val
Leu Lys Asn 85 90 95Tyr Gln Asp Met Val Val Glu Gly Cys Gly Cys Arg
100 105
* * * * *