TWI328036B - Process for producing c-termial histidine tagged hoxb4 and the use thereof in expanding stem cells - Google Patents

Description

139. The invention relates to the improvement of the recombination by constructing a DNA construct for producing a histidine-tagged HOXB4 recombinant protein (TAT-HOXB4H) at the c-terminus. A protein purification efficiency method wherein the TAT-HOXB4H contains 6 histidine labels at the C-terminus. The present invention is also further directed to a method and composition for using the produced TAT-HOXB4H recombinant protein for in vitro stem cell proliferation.

[Prior Art] Umbilical cord blood (UCB) refers to blood remaining in the umbilical cord and placenta at the time of birth. Umbilical cord blood is rich in "zero-year-old" primitive stem cells, which have the same function as bone marrow and are the main source of blood and immune systems. Cord blood stem cells are also called totipotent cells. Because they are similar to embryos, they are "young" and less differentiated cells. They can be developed into different types of cells or tissues. They can be used to treat cancer, blood diseases, and even to innate Metabolic diseases, etc. The number and quality of cord blood stem cells are superior to those of other stem cells, because the concentration of hematopoietic stem cells and mother cells in cord blood is 10-20 times higher than that in bone marrow, and its proliferation ability is also high. V· _ Furthermore, because the tb blood hematopoietic stem cell tb is less mature (immature), its T cell's ability to recognize "self" and "non-self" is more mature than bone marrow or peripheral blood. Hematopoietic stem cells are poor, so it can reduce the incidence of graft versus host disease (GVHD) and rejection, and can also increase the transplantation of minority groups.

5 TBH02-P424-TW 1328036 Opportunity. In addition, cord blood stem cell pairing requirements are relatively low. Bone marrow transplantation between non-relatives requires six white blood cell antigens (HLA) to be matched. Umbilical cord blood stem cell transplantation can be transplanted as long as five or even four antigens are identical. Umbilical cord blood stem cells are collected in limited quantities and are acceptable for babies' childhood use, but may be slightly inadequate if used in adults. Dr. Guy Sauvageau of the University of Montreal in Canada has explored the limitation of the number of stem cells in the bone marrow.

It is important to show that the homeobox gene 7/0X54 regulates the self-renewal of hematopoietic stem cells and maintains its cluster size in the bone marrow. The earliest evidence that genes are expressed in blood cells is confirmed by the cell lines of humans and mice. Some T/οχ genes have a wide range of expression in different cell morphology, and some genes are only activated in specific cells. For example, 'eight members of the human ulnar group will behave at the beginning of red blood cell development, and some // Chiyou 5 genes include //0^4 and//(9X57 will also be expressed in T cells and B cells. Sauvageau et al. It has been confirmed that nine if CUC4 genes, eight //0X5 genes and four //OXC genes are expressed in CD34+ bone marrow cells, which are again in the cell population of CD34+ in 9×52, then and //0X4. The erythrocyte primordial cells performed the most - in addition, the experimental results also showed that there was no //0 gene or a small amount of ZfOZ gene expression in the CD34-cell population. Therefore, the r H0XB4" protein has been most commonly used as a living body. An effective stimulant for the proliferation of exogenous hematopoietic stem cells (HSC). 0 The recombinant "Η0ΧΒ4" protein produced by genetic engineering is used to terminate the "Η0ΧΒ4" protein in order to allow the protein to enter the cell.

TBH02-P424-TW 1328036 is a protein sequence of TAT that allows the "HOXB4" protein to be folded into an active conformation with the help of Hsp90 after the "HOXB4" protein enters the cell. Further, a histidine acid label containing 6 histidine acids (His-6) is contained at the N-terminus thereof to facilitate purification and recovery of the recombinant protein. It has also been demonstrated that the recombinant "TAT-HOXB4" recombinant protein can promote hematopoietic stem cell proliferation by 2-6 fold (Amsellem, S. et al.

Mei/zczwe 9, 1423-1427, 2003; and Krosl, J. et al., 1428-1432, 2003). However, the purification yield of the TAT-H0XB4 recombinant Φ protein from the E. coli host is low, so in order to obtain a sufficient amount of TAT-HOXB4 recombinant protein for stem cell proliferation, a large number of E. coli expression cells must be cultured, and more complicated use is required. Purification step.

Thus, the present invention provides a solution for improving the recovery yield of TAT-HOXB4 recombinant protein, mainly by constructing a construct for producing a C-terminal histidine-containing labeled HOXB4 (TAT-HOXB4H) recombinant protein. Wherein the C-terminus of the recombinant TAT-HOXB4 protein expressed has a marker of 6 histidine residues (His-6), thereby improving the purification efficiency of the recombinant protein. The purified and stem cell proliferation experimental data of the present example shows that the TAT-HOXB4H recombinant protein produced by the method of the present invention produces '3-5 times higher purification efficiency (and purified yield) than the original TAT-HOXB4 recombinant protein. And has the ability to promote stem cell proliferation comparable to the TAT-HOXB4 recombinant protein. SUMMARY OF THE INVENTION In one aspect, the present invention relates to a preparation of a C-terminal containing six histidine acids.

7 TBH02-P424-TW 1328036 A method for the HOXB4 recombinant protein (TAT-HOXB4H), which comprises constructing a DNA construct comprising six histidine-encoding fragments at the C-terminus of the HOXB4 open reading frame (ORF); The resulting construct is transformed into a host cell to express the TAT-HOXB4H recombinant protein; and the recombinant protein is purified. The terms "expression carrier", "polynucleotide vector", "DNA construct" and "carrier construct" are used interchangeably herein to mean any construct for gene transfer, as in the art. People understand.

In a specific aspect of the present invention, a HOXB4 open reading frame containing 6 histidine-encoding fragments is introduced into the C-terminus by PCR, and the DNA fragment is cloned into an appropriate host cell. Performed in the middle. In another embodiment, a vector capable of expressing a recombinant protein of TAT-HOXB4 is used as a template, and a coding sequence of four histidines is more than one of the original ΤΑΤ-ίίΟΧΒ4 by PCR. In the terminal nucleotide sequence, the final expression of the ΤΑΤ-ΗΟΧΒ4Η recombinant protein contains six histidine labels at the C-terminus. After the transformation of the construct into Escherichia coli, the recombinant protein of ΤΑΤ-ΗΟΧΒ4Η was obtained, and the purified yield was 3-5 times higher than that of the original ΤΑΤ-ΗΟΧΒ4 recombinant protein. The recombinant protein of ΤΑΤ-ΗΟΧΒ4Η obtained by the method of the present invention can also be used for stem cell proliferation. It has been proved by experiments that the umbilical cord blood stem cells can be proliferated 6 times in vitro, which is equivalent to the original ΤΑΤ-ΗΟΧΒ4 recombinant protein, indicating that the four histidine acids are introduced into the ΤΑΤ-ΗΟΧΒ4 recombinant protein C- according to the method of the present invention. The modified ΤΑΤ-ΗΟΧΒ4Η recombinant protein prepared at the end can not only increase the purification efficiency of the host cell (and the yield after purification), but also can be effectively used for stem cell proliferation.

TBH02-P424-TW 1328036 Therefore, another aspect of the present invention relates to a stem cell proliferation culture comprising the novel TAT-HOXB4H recombinant protein produced in stem cell proliferation. In a specific aspect of the invention, the stem cell line for boosting is cord blood stem cells. In another specific aspect, the 'supplied stem cell line cord blood hematopoietic stem cells. • The term “stem cell” refers to a cell that has the ability to proliferate, self-repair, and mass-produce differentiated progeny. Stem cells have the ability to differentiate or transform into other cells, usually from the early stages of embryonic development, and can be transformed into specific tissues and organs under appropriate environmental conditions. [Embodiment] The following examples are intended to illustrate the technical effects of the present invention, but are not intended to limit the present invention. Any equivalent changes and modifications made in accordance with the invention are within the scope of the invention. Example 1. Construction of plastid PTAT-HOXB4H

The arm pTAT-HOXB4 (Nature Medicine 9, 1428-1432 (2003)) was amplified by PCR reaction using the following primers: 5'-ctccatggctatgagttcttttttg-3' and «.**· 5 ' -atgatgatgatgatgatggagcgcgcgg- 3 ', and a fragment containing four additional histidine residues at the C-terminus of the HOXB4 open reading frame (ORF) (see Figure 2). The amplified fragment was further amplified by PCR reaction using the following primer pairs: 5'-ctccatggctatgagttcttttttg-3' and 5'-cagaattcctaatgatgatgatgatga-3'', and one segment at the 3' end

TBH02-P424-TW 1328036

And month ’/p :t hand I ’ f.· ':" . A newly generated fragment of the EcoRI cut. Then limit this fragment

Ncol is cut with EcoRI and subcultured to the skeleton of the original PTAT-HOXB4 plastid cut by the same enzyme (the Ncol and EcoRI cleavage on it) to obtain the novel plastid pTAT- HOXB4H, the DNA sequence thereof is shown in SEQ ID NO: 1 of the Sequence Listing. • Example 2. Purification and yield comparison of TAT-HOXB4H recombinant protein with TAT-HOXB4 recombinant protein The PTAT-HOXB4 (or pTAT-HOXB4H) vector was transformed into E. coli B12 (DE3) pLysS (Novagen). The transformed cells were grown overnight at 37 °C. The overnight culture was diluted to a starting OD6Q 0.05 value of 0.05 and grown to a 00_value of 0.5 at 37 ° C followed by 1 mM isopropylthio-β-D-galactose (IPTG) at 37°. Induction was performed for 3 hours under C with vigorous shaking. After the induction, the cells were collected by centrifugation and resuspended in buffer A (8 M urea, 20 mM HEPES, 0.5 mMDTT and 100 mM NaCl ρ Η 8·0). The cell suspension was dialed three times through a French press, and the lysate was clarified by centrifugation at 20,000 x g for 30 minutes at 4 °C. The Θ supernatant was adjusted to 10 mM imidazole and applied to a HisTrap chelating column (Amersham Pharmacia). The bound protein was eluted with imidazole prepared in buffer A at 50, 100 and 250 mM. The fraction containing HOXB4 (or ΗΌΧΒ4Η) was orally applied to a MonoSP column in the presence of buffer B (4M urea, 20 mM HEPES and 50 mM NaCl ρΗ6·5), and eluted with 1.5 M NaCl and 20 mM HEPES (ρΗ8·0). It was placed on a PD-10 Sephadex G-25 column to remove salt. The HOXB4 (or HOXB4H) protein was eluted from the PD-10 Sephadex G-25 column in PBS with a purity of >99%. Will dissolve all

TBH02-P424-TW 1328036 was added to 1% BSA and 10% glycerol, dispensed and snap frozen at -80 °C. The electrophoresis results shown in Figure 3 show that the C-terminal new product produced by the method of the present invention: 4 TERN-HOXB4H recombinant protein of histidine, the yield of the recombinant protein in the E. coli host is lower than that of the original TAT-HOXB4 recombinant protein. Increase by about 3-5 times. The yield of recombinant protein was quantified by Bradford assay. The OD595 was used to map the known concentration of BSA, and the standard curve was used to calculate the protein content in the sample by interpolation. The yield of TAT-HOXB4H recombinant protein was found to be 5.5 mg/L, while the yield of TAT-HOXB4 recombinant protein was 1.2 mg/L. Example 3. Ability of TAT-H0XB4H recombinant protein for gold stem cell proliferation

The red blood cells in the human cord blood were removed by dissolution at 0.8 ° C in ammonium sulfate (pH 7.0) containing 0.1 ° / 〇 sodium hydrogencarbonate at 4 ° C. After the collected antibodies and the specific antibodies bound to the magnetic beads are co-cultured with the cells, the magnetic cell separation procedure is performed by Stemsep column separation to collect the CD34+ cells, and the part showing the mature human white blood cells is purified. Cells in stem cell culture medium (DMEM containing 10% FCS, 5

Jig/ml interleukin-3 '10 ng/ml interleukin _6, 100 ng/ml STF, penicillin 100 units/ml and 100 pg/ml streptomycin) for 2 days, then placed at 15 nM The cells were cultured for 4 days in H0XB4 (or H0XB4H) or 1% BSA stem cell culture medium. On the third day of culture (on the day of treatment, the day of the treatment), 4 <1〇5 cells/ml was resuspended in the stem cell medium supplemented with BSA or H0XB4 (or HOXB4H). Then add fresh BSA every 3 hours or

TBH02-P424-TW 1328036 HOXB4 (or HOXB4H) protein (containing 50% of the initial protein amount dissolved in • 5% total medium). After 12 hours, FCS and cytokines were added to correct the correct amount contained in the medium at a dilution of 20%. After 24 hours, the cells were resuspended in fresh medium containing the protein of interest. On the 0th, 2nd and 4th day of treatment, the amount of stem cells was counted by the ISHAGE method (CD45/CD34 staining plus forward and side scatter light). From the results of Fig. 4, it was confirmed that the TAT-HOXB4H recombinant protein of the present invention can proliferate about 6 times for cord blood stem cells, and the potency is comparable to the conventional TAT-HOXB4 recombinant protein. It is indicated that the TAT-HOXB4H recombinant protein containing the additional 4 histidines at the C-terminus according to the present invention has no effect on the stem cell proliferation ability of the original TAT-HOXB4 recombinant protein, and will be combined with TAT-HOXB4H. Protein co-culture of proliferating human CD34+ cord blood stem cells (5000 cells per mouse) with 5xl04

CD34·radiation-assisted helper cells were injected together into radiation-irradiated (2.5 Gy) NOD-LtSz-scid/scid (NOD-SCID) mice. Seven weeks after transplantation, the amount of human CD45+ cells in blood cells taken from the transplanted animals was analyzed by flow cytometry. As a result, the stem cells treated with the TAT-H0XB4H protein of the present invention can be mixed into the bone marrow of the NOD-SCID mouse compared to the BLA-treated cord blood stem cells of the same JL amount, and are comparable in the peripheral white blood cells of the mouse. Containing 0.05 to 0.07% of human CD45-positive white blood cells. This indicates that these proliferating stem cells still retain pluripotency. Proved by the yield analysis and stem cell proliferation test results shown above

12 TBH02-P424-TW 1328036 Sequence Listing <110> Taiwan Advanced Advanced Biotechnology Co., Ltd. <120> C-terminal method for producing histidine-tagged HOXB4 recombinant protein and its use for stem cell proliferation< 160>1

<210> 1 <211> 1050 <212>DNA <213>Artificial sequence <220><223> is DNA sequence of pTAT-HOXB4H construct <400> GTTTCCTCTA GAATAATTTT GTTTACTTTA AGAAGGAGAT 40 ATACATATGC GGGGTTCTCA TCATCATCAT CATCATGGTA 80 Met(start) His6

TGGCTAGCAT GACTGGTGGA CAGCAAATGG GTCGGGATCT 120 GTACGACGAT GACGATAAGG ATCGATGGGG ATCCAAGCTT 160 GGCTACGGCC GCAAGAAACG CCGCCAGCGC CGCCGCGGTG 200 TAT-seq. GATCCACCAT GTCCGGCTAT CCATATGACG TCCCAGACTA 240 HA-tag TGCTGGCTCC ATGGCTATGA GTTCTTmT GATCAACTCA 280 Ncol AACTATGTCG ACCCCAAGTT CCCTCCATGC GAGGAATATT 320 CACAGAGCGA TTACCTACCC AGCGACCACT CGCCCGGGTA 360 CTACGCCGGC GGCCAGAGGC GAGAGAGCAG CTTCCAGCCG 400 GAGGCGGGCT TCGGGCGGCG CGCGGCGTGC ACCGTGCAGC 440 GCIACGCGGC CTGCCGGGAC CCTGGGCCCC CGCCGCCTCC 480 i 1328036

CCTCGGGCTC CTGCGCCGCC ACCCGCCGGG GCCCTCCTCC 560 CGGAGCCCGG CCAGCGCTGC GAGGCGGTCA GCAGCAGCCC 600 CCCGCCGCCT CCCTGCGCCC AGAACCCCCT GCACCCCAGC 640 CCGTCCCACT CCGCGTGCAA AGAGCCCGTC GTCTACCCCT 680 GGATGCGCAA AGTTCACGTG AGCACGGIAA ACCeCAATTA 720 CGCCGGCGGG GAGCCCAAGC GCTCTCGGAC CGCCTACACG 760 CGCCAGCAGG TCTTGGAGCT GGAGAAGGAA TTTCACIACA 800 ACCGCTACCT GACACGGCGC CGGAGGGTGG AGATCGCCCA 840 CGCGCTCTGC CTCTCCGAGC GCCAGATCAA GATCTGGTTC 880 CAGAACCGGC GCATGAAGTG GAAAAAAGAC CACAAGTTGC 920 CCAACACCAA GATCCGCTCG GGTGGTGCGG CAGGCTCAGC 960 CGGAGGGCCC CCTGGCCGGC CCAATGGAGG CCCCCGCGCG 1000 CTCCATCATC ATCATCATCA TTAGGAATTC AAAGCTTGAT 1040 C-terminal Histidine tag EcoRI CCGGCTGCTA 1050

2

Claims (1)

X. The scope of application for patents: 99. ϋ. 11 _j£---η年月 S'充充@'本1. A H0XB4 recombinant protein (TAT-H0XB4H) containing 6 histidine tags at the C-terminus. The method comprises the following steps: (1) constructing a DNA construct comprising a 6 histidine-encoding fragment at the C-terminus of the H0XB4 open reading frame (ORF), which has the DNA sequence set forth in SEQ ID NO: (2) transforming the construct obtained in the step (1) into the E. coli host cell to express the TAT-HOXB4H recombinant protein; and (3) the recombinant E. coli host cell lysate obtained from the step (2); The TAT-HOXB4H recombinant protein was purified. 2. The method according to claim 1, wherein the DNA construct system introduces an additional four histidine acid coding sequences into the C-terminus of the TAT-HOXB4 open reading frame (ORF) by a PCR method. 3. A DNA construct for increasing the production of recombinant protein of TAT-HOXB4H, characterized in that the C-terminus of the open reading frame of TAT-HOXB4 has been introduced with 6 histidine tags and has SEQ ID NO: 1 DNA sequence. 4. A medium for in vitro proliferation of stem cells, comprising 5-100 nM of TAT-HOXB4H recombinant protein prepared according to the method of claim 1 of the patent application, and cell nutrients, cytokines and cells suitable for stem cell proliferation antibiotic. D. BH02-P424-TW 1328036 5. The medium according to claim 4, wherein the stem cells are cord blood stem cells. 6. The medium according to claim 4, wherein the stem cells are hematopoietic stem cells. 2 TBH02-P424-TW (§) 1328036 XI. Schema: HA tag, TAT ® 0Q. \ ΗΘΚΒ4 Χ-'ιΛί:--': t- '·' -:-;λ··»« HieT^p of.prinier PTAT-HOXB4 A Γ 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 引 L TBH02-P424-TW 1328036 Μ 66.4 55.6 47.5 HOXB4/HOXB4H 34.6 27 2· TBH02-P424-TW 1328036 □ TAT-HOXB4 ▲ TAT-HOXB4H ♦ BSA TBH02-P424-TW