TWI756518B - Mutated recombinant hoxb4 proteins, method for manufacturing same, and use thereof - Google Patents

Abstract

The present disclosure relates to the mutated recombinant HOXB4 proteins, including at least one mutation at three specific positions within the DNA binding domain. The mutation enhances DNA binding ability and potency of inducing hematopoietic stem cell expansion compared to wild type HOXB4.

Description

Production method and use of mutant HOXB4 recombinant protein

The present invention relates to the production method and application of mutant HOXB4 recombinant protein. The mutant obtained by point mutation of three specific positions in the DNA binding region can improve the binding ability with DNA and proliferate hematopoietic stem cells. Effect.

Umbilical cord blood refers to a part of fetal blood circulation. During pregnancy, the placenta exchanges oxygen and nutrients with the mother, and the blood remains in the umbilical cord and placenta when the baby is born. Umbilical cord blood is rich in primitive stem cells, and its function is equivalent to that of bone marrow. It is the main source of the body's blood production and immune system.

Hematopoietic stem cells are undifferentiated blood cells that have the ability to self-renew and manufacture blood and the immune system, such as red blood cells that transport oxygen, white blood cells that fight infection, platelets that help blood clot, and lymphocytes that produce antibodies. At present, in addition to the treatment of many blood diseases, immune deficiencies and tumors, hematopoietic stem cell transplantation is widely used to rebuild hematopoietic and immune functions damaged by radiation therapy and chemotherapy, and to regenerate new blood and the immune system to prolong the life of the patient.

The concentration of hematopoietic stem cells and precursor cells in umbilical cord blood is 10-20 times higher than that in bone marrow, and the proliferation ability is also better. In addition, the pairing requirements are low. Non-relative bone marrow transplantation requires all six white blood cell antigens to be met, and umbilical cord blood stem cell transplantation only requires four or five antigens to be the same.

However, the number of umbilical cord blood stem cells that can be collected is limited, which is acceptable if used in early childhood, and slightly insufficient if used in adults. Previous studies have shown that the homeobox gene HOXB4 is very important for the regulation of hematopoietic stem cell self-renewal and maintains its large clustering in the bone marrow. little.

Dr. Guy Sauvageau of the University of Montreal, Canada, and his team have studied the limited number of stem cells in the bone marrow. Expressed in blood cells. Among them, HOXB4 protein has been most commonly used as a potent stimulator of hematopoietic stem cell (HSC) proliferation in vitro.

In order to enhance the effect of HOXB4 protein as a stimulator of hematopoietic stem cell proliferation, several studies have been devoted to improving the stability of the protein using point mutation or sequence fragment deletion techniques. For example, US Patent Publication No. 8039436B2 discloses the substitution of at least one of the 6th, 7th, 23rd and 28th amino acids of the HOXB4 amino acid sequence with a lipophilic amino acid, and the deletion of the amino acid sequence 31 to 35. All amino acids help reduce the effect of the protein being hydrolyzed by the proteasome through the labeling of ubiquitin. The case of US Patent Publication No. 9783783B2 revealed that point mutation of the LEXE motif on HOXB4 (amino acid sequence 175, 176 and 178) can effectively reduce the proteolysis mediated by CUL4 . However, no studies have revealed that the mutant HOXB4 protein has better deoxyribonucleic acid (DNA) binding ability.

HOXB4 is also a DNA-binding protein, which contains a DNA-binding region in its structure. Dr. Ye Chen from Hongguang University of Science and Technology has previously been entrusted to solve the structure of the DNA-binding region of HOXB4 by X-ray crystallography (Figure 1). Its structure is similar to that of HOXB1 protein named 1B72 in the Protein Data Bank (Figure 2). According to the simulated three-dimensional structure of the DNA binding region and DNA binding of HOXB4 protein, the 47th, 48th and 59th amino acids located in the binding region sequence play important roles in DNA binding (Figure 3). These three positions are arginine (Arginine, R), glutamic acid (Glutamine, Q) and lysine (Lysine, K), which correspond to the 204th, 205th and 216th amines in the full-length sequence of HOXB4, respectively. Base acid, for consistency, R204, Q205 and K216 will be used in subsequent descriptions.

Therefore, the present invention uses genetic engineering technology to target the above-mentioned three specific positions in the DNA binding region of the HOXB4 protein to carry out amino acid mutation, which is carried out by polymerase chain reaction. plastid point mutation, which in turn alters the amino acids in its protein phase. The DNA binding ability of the single mutation point recombinant protein and the double mutation point recombinant protein prepared by the present invention is improved, and the stem cell proliferation ability of the double mutation point recombinant protein is better than that of the wild-type HOXB4 protein.

Based on the above purpose, the present invention is aimed at the above-mentioned three specific positions of the DNA binding region of the HOXB4 protein, performing amino acid mutation, and performing plastid point mutation by polymerase chain reaction, so as to obtain a protein compared to the wild-type HOXB4 protein. , has better binding ability with DNA molecules, and better stem cell proliferation ability.

Thus, one aspect of the present invention relates to a mutant homeobox B4 (HOXB4) protein comprising the following amino acid sequence: the amino acid sequence represented by SEQ ID NO: 2 or an amino acid having at least 95% identity therewith In the sequence, the amino acid corresponding to at least one of the 204th, 205th and 216th positions of the amino acid sequence represented by SEQ ID NO: 2 is mutated, and the amino acid sequence comprising the amino acid sequence represented by SEQ ID NO: 2 Or the mutant HOXB4 protein has a higher binding ability to DNA than its wild-type HOXB4 protein having an amino acid sequence of at least 95% identity.

In some embodiments of the present invention, the above-mentioned protein, wherein the amino acid corresponding to at least one of positions 204, 205 and 216 of the amino acid sequence represented by SEQ ID NO: 2 is mutated to neutral or Positively charged amino acid.

In some embodiments of the present invention, the above-mentioned HOXB4 protein includes at least one of the following mutations: the amino acid corresponding to the 204th position of the amino acid sequence represented by SEQ ID NO: 2 is mutated to histidine (Histidine; His; H) or lysine (Lysine; Lys; K); the amino acid corresponding to the 205th position of the amino acid sequence represented by SEQ ID NO: 2 is mutated to histidine (H) or Arginine (Arginine; Arg; R); and the amino acid corresponding to the 216th position of the amino acid sequence represented by SEQ ID NO: 2 are mutated For histidine (H) or arginine (R).

In other embodiments of the present invention, the above-mentioned HOXB4 protein, wherein the amino acids corresponding to the 205th and 216th positions of the amino acid sequence represented by SEQ ID NO: 2 are mutated into histidine (H) and Arginine (R).

Another aspect of the present invention relates to a recombinant protein comprising the above-mentioned HOXB4 protein and a cell-penetrating peptide.

In some embodiments of the present invention, the recombinant protein, wherein the cell-penetrating peptide is a trans-activator of transcription (TAT).

In other specific embodiments of the present invention, the recombinant protein, wherein the transcription activator comprises the sequence of SEQ ID NO: 4.

Yet another aspect of the present invention relates to a method for cell expansion, comprising administering an effective amount of the HOXB4 protein of claim 1 for promoting cell expansion to target cells to expand the cells.

In some embodiments of the present invention, the above-mentioned method is to administer an effective amount of HOXB4 protein to promote cell expansion to target cells in vitro.

In some embodiments of the present invention, the above-mentioned method, wherein the target cell line is a precursor cell hematopoietic stem cell, and/or a cell induced by the differentiation.

In some embodiments of the present invention, the above method, wherein the target cell is a hematopoietic stem cell.

In some embodiments of the present invention, in the above method, the source of the hematopoietic stem cells is umbilical cord blood.

In some embodiments of the present invention, in the above method, the source of the hematopoietic stem cells is peripheral blood.

In some embodiments of the present invention, in the above method, the source of the hematopoietic stem cells is bone marrow.

Fig. 1 The structure of the DNA-binding region of HOXB4 protein solved by X-ray crystallography.

Figure 2. Structure diagram of HOXB1 protein binding to Pbx1 and DNA (Protein data bank ID: 1B72)

Fig. 3 The simulated three-dimensional structure diagram of the DNA-binding region of HOXB4 protein and DNA-binding. The 47th Arginine (R), the 48th Glutamine (Q) and the 59th Lysine (K) in the binding region sequence play an important role in binding with DNA.

Figure 4 is the amino acid sequence of the wild-type recombinant protein of TAT-HOXB4, in which the full-length HOXB4 sequence is indicated by the bottom line, the DNA binding region is indicated in bold, and the amino acid position to be mutated is indicated in reverse white.

Figure 5 is the target DNA sequence of pTAT-HA-HOXB4 plastid, in which the DNA bonding region is marked in bold, and the coding sequence position to be mutated is marked in reverse white.

Fig. 6 The wild-type/mutant-type TAT-HOXB4 recombinant protein can be induced and produced by IPTG in the E. coli expression system, and the arrow indicates the electrophoresis position of the target protein.

Figure 7 Purified mutant TAT-HOXB4 recombinant protein.

Figure 8 shows the measurement of the binding of wild-type and mutant TAT-HOXB4 proteins to DNA by surface plasmon resonance biosensors.

Figure 9 presents the results of comparing wild-type and mutant TAT-HOXB4 proliferating hematopoietic stem cells in vitro, expressed as a measure of the number of CD34+ cells. The control group was treated with bovine serum albumin (BSA).

A "stem cell" is a pluripotent or multipotent cell with the ability to self-renew and differentiate into different types of cells. Stem cells naturally exist in animals and provide various sources of cells needed by animals through their differentiation potential. When stem cells divide, their progeny cells still have stem cell characteristics, or they can go down the pathway of differentiation and become mature adult cells.

A "hematopoietic" cell refers to a cell involved in the process of hematopoiesis (ie, the process by which precursor cells form mature blood cells). In adults, hematopoiesis occurs in the bone marrow. During different developmental stages early in development, hematopoiesis occurs at different sites; primitive blood cells appear in the yolk sac, and later, blood cells form in the liver, spleen, and bone marrow. Hematopoiesis is a process that undergoes complex regulation, including hormones (eg, erythropoietin), growth factors (eg, colony-stimulating factors), and cytokines (eg, interleukins).

The present invention is aimed at the above-mentioned three specific positions of the DNA bonding region of HOXB4, and carries out amino acid mutation, which mainly uses genetic engineering technology to carry out plastid point mutation by polymerase chain reaction, thereby changing its protein stage. of amino acids.

The following examples are used to illustrate the effects that can be achieved by the technical content of the present invention, and are not intended to limit the present invention. Any equivalent changes and modifications made according to the present invention, within the scope and spirit of the invention, fall within the scope of the application scope of the present invention.

Example

Example 1 Construction of point mutant plastids

Using the pTAT-HA-HOXB4 plastid as a template (as shown in Figure 5), the polymerase chain reaction (Polymerase Chain Reaction, PCR) was used to carry out site-directed mutagenesis (site-directed mutagenesis) amplification for specific positions R204, Q205 and K216. increase reaction. Two amino acid mutations were carried out with the following designed primers (as shown in Table 1): Histidine(H)/Lysine(K) or H/Arginine(R). After colonization and sequencing confirmation, the obtained new The clones were named pTAT-HA-HOXB4_R204H, pTAT-HA-HOXB4_R204K, pTAT-HA-HOXB4_Q205H, pTAT-HA-HOXB4_Q205R, pTAT-HA-HOXB4_K216H and pTAT-HA-HOXB4_K216R, respectively. For double point mutation, pTAT-HA-HOXB4_Q205H was used as a template, and the K216R primer set in Table 1 was used for site-directed mutagenesis amplification reaction, and the new plastid was named pTAT-HA-HOXB4_Q205H_K216R.

Example 2 Comparison of expression and purification of wild-type and point mutant TAT-HOXB4 recombinant proteins

(1) pTAT-HA-HOXB4_R204H; (2) pTAT-HA-HOXB4_R204K; (3) pTAT-HA-HOXB4_Q205H; (4) pTAT-HA-HOXB4_Q205R; (5) pTAT-HA-HOXB4_K216H; (6) pTAT - HA-HOXB4_K216R; pTAT-HA-HOXB4_Q205H_K216R were transformed into E. coli strain BL21(DE3) pLysS (Novagen), respectively. The transformed E. coli cells were grown overnight at 37°C with 150 rpm rotating shaking. The overnight broth was diluted 1:50 to fresh broth, starting with an _OD600 value of approximately 0.05, and grown at 37°C with 200 rpm rotational shaking until an _OD600 value of 0.7-0.8, followed by 1 mM isopropylthio -bD-galactose (IPTG) induced protein expression for 3 hours at 37°C with 200 rpm rotary shaking.

After induction, cells were collected by centrifugation and re-lysed with buffer A (8M urea, 20 mM HEPES, 100 mM NaCl, pH 8.0). The cell suspension was extruded three times with a high pressure differential French Press, and the cell lysate was clarified by centrifugation at 18,000 rpm at 4° C. for 30 minutes, and the supernatant was collected. The supernatant was adjusted to a buffer containing 10 mM imidazole, and the samples were applied to HisTrap affinity columns (GE Healthcare), and the bound proteins were quantified in buffers containing 50, 100, 150, 300, 500 and 1000 mM imidazole. Buffer A for elution. Elution samples containing mutant HOXB4 were collected, applied to a HiTrap SP cation exchange chromatography column, and eluted using a gradient in buffer B (20 mM HEPES, 1 M NaCl, pH 8.0) without imidazole and urea. Elution samples containing mutant HOXB4 of the target protein were collected and the protein was refolded by dialysis. The refolded protein samples were aliquoted, snap-frozen in liquid nitrogen, and stored at -80°C.

The theoretical molecular weight and isoelectric point of the protein expressed in the plastids after point mutation are very little different from those of wild-type HOXB4 (HOXB4_WT), as shown in Table 2.

Example 3 The ability of wild-type and point mutant TAT-HOXB4 recombinant proteins to bind to deoxyribonucleic acid DNA

Using Surface plasmon resonance (SPR) surface plasmon resonance biosensor to measure the dynamic binding and dissociation process between HOXB4 protein and DNA HOXB4 binding consensus DNA sequence (binding consensus DNA sequence: 5'-CTGCGATGATTGATGACCGC-3'.), compare Wild type HOXB4 (SEQ ID NO: 32) and mutant HOXB4 (containing single mutations R204H (SEQ ID NO: 34), R204K (SEQ ID NO: 36), Q205H (SEQ ID NO: 38), Q205R (SEQ ID NO: 40), K216H (SEQ ID NO: 42) , K216R (SEQ ID NO: 44) and double point mutation Q205H_K216R (SEQ ID NO: 46)) ability to bind to DNA. From the data in Figure 8, it can be seen that except the binding ability of HOXB4_K216H is similar to that of wild-type HOXB4, the binding ability of other single-mutated HOXB4 recombinant proteins to DNA is enhanced by 1.39-1.89 times, while the double-point mutant recombinant protein HOXB4_Q205H_K216R has a binding ability to DNA. The binding capacity is 50 times that of wild-type HOXB4. The binding ability of recombinant protein to DNA measured by this method shows that HOXB4_R204K is the strongest single mutant, followed by HOXB4_Q205R. The double-point mutation recombinant protein HOXB4_Q205H_K216R has far better binding ability to DNA than any single point mutation recombinant protein and wild-type HOXB4.

Example 4 The ability of wild-type and point mutant TAT-HOXB4 recombinant proteins to proliferate hematopoietic stem cells in vitro

Red blood cells from human umbilical cord blood were prepared at 4°C with 0.1% Sodium bicarbonate was dissolved in 0.83% ammonium chloride (pH 7.0) and removed. After the collected leukocyte fraction was co-incubated with the cells with specific antibodies bound to magnetic beads, a magnetic cell separation procedure was performed by Stemsep column separation to collect CD34+ cells and remove the fraction representing mature human leukocytes. Cells of a specific density were cultured in a six-well plate, and a specific concentration of BSA or wild-type or mutant TAT-HOXB4 recombinant protein was added in the morning and evening for 4 consecutive days. On the 5th day, flow (LIN/CD34 staining plus positive and side scatter light) to count the number of stem cells and analyze.

The results in FIG. 9 prove that the proliferative ability of the single-point mutant TAT-HOXB4 recombinant protein prepared according to the present invention on umbilical cord blood stem cells is TAT-HOXB4_Q205H the best, comparable to wild-type TAT-HOXB4, followed by TAT-HOXB4_K216R . According to this result, the recombinant protein TAT-HOXB4_Q205H_K216R, which was further mutated by double point, was better than the above two, and slightly better than wild-type TAT-HOXB4 for hematopoietic stem cells.

Based on the above, the DNA binding ability and stem cell proliferation experiment data in the examples of this case show that the single mutation point recombinant protein prepared by the present invention has a 1.4-1.9-fold increase in the binding ability to DNA, and HOXB4_Q205H has a recombinant protein with wild-type HOXB4. Equivalent stem cell proliferative capacity. According to the analysis data of the stem cell proliferation ability of the recombinant protein with a single mutation point, the two-point amino acid mutation HOXB4_Q205H_K216R was selected at the Q205 and K216 positions. The mutant double point mutation recombinant protein HOXB4_Q205H_K216R recombinant protein prepared by the method of the present invention can also be used for stem cell proliferation, and the stem cell proliferation ability is better than wild-type HOXB4.

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Claims (11)

A mutant homeobox B4 (HOXB4) protein comprising the following amino acid sequence: in the amino acid sequence represented by SEQ ID NO: 2, corresponding to positions 204, 205 and 216 of the amino acid sequence represented by SEQ ID NO: 2 The amino acid in at least one position is substituted with a neutral or positively charged amino acid, and compared with the parent HOXB4 protein comprising the amino acid sequence represented by SEQ ID NO: 2, the mutant HOXB4 protein has Higher binding capacity to DNA; wherein the neutral or positively charged amino acid is selected from the group consisting of histidine, lysine and arginine. The protein according to claim 1, which comprises at least one of the following substitutions: a. The amino acid at the position corresponding to the 204th position of the amino acid sequence represented by SEQ ID NO: 2 is substituted with histidine or lysine acid; b. the amino acid corresponding to the 205th position of the amino acid sequence represented by SEQ ID NO: 2 is substituted with histidine or arginine; and c. corresponding to the amino group represented by SEQ ID NO: 2 The amino acid at position 216 of the acid sequence was substituted with arginine. The protein according to claim 1, wherein the amino acids corresponding to positions 205 and 216 of the amino acid sequence represented by SEQ ID NO: 2 are substituted with histidine and arginine, respectively. A recombinant protein comprises the protein of claim 1 and a cell-penetrating peptide. The recombinant protein of claim 4, wherein the cell-penetrating peptide is a trans-activator of transcription (TAT). The recombinant protein according to claim 5, wherein the transcription activator comprises the sequence of SEQ ID NO: 4. A method for cell expansion, comprising administering an effective amount of the protein of claim 1 for promoting cell expansion to a target cell in vitro to expand the cell; wherein the target cell line is precursor cells, hematopoietic stem cells, and/or Cells induced by these differentiations; wherein the protein corresponding to the amino acid at the 205th position of the amino acid sequence represented by SEQ ID NO: 2 is substituted with histidine and the amino acid at the 216th position It is substituted with arginine. The method of claim 7, wherein the target cell is a hematopoietic stem cell. The method of claim 8, wherein the source of the hematopoietic stem cells is umbilical cord blood. The method of claim 8, wherein the source of the hematopoietic stem cells is peripheral blood. The method of claim 8, wherein the source of the hematopoietic stem cells is bone marrow.

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