TWI756518B - Mutated recombinant hoxb4 proteins, method for manufacturing same, and use thereof - Google Patents
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Abstract
Description
本發明係關於突變型HOXB4重組蛋白質之製造方法及用途,對去氧核醣核酸結合區域的三個特定位置進行點突變所得之突變構體,可提高與去氧核醣核酸的結合能力以及增生造血幹細胞的效果。 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.
臍帶血係指胎兒血液循環的一部分,在母體懷孕期間,藉由胎盤與母體進行氧氣及養分交換,於嬰兒出生時,留存於臍帶與胎盤的血。臍帶血富含原始幹細胞,其功用與骨髓相當,是人體製造血液及免疫系統的主要來源。 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.
造血幹細胞是未分化的血液細胞,具有自我更新的能力,並且有製造血液與免疫系統的能力,如運輸氧氣的紅血球、抵抗感染的白血球、幫助凝血的血小板、以及產生抗體的淋巴球等。目前,造血幹細胞除了用來治療許多血液疾病、免疫缺陷與腫瘤等疾病外,造血幹細胞移植被廣泛應用於為重建因放射治療、化學治療而損傷之造血、免疫機能,可再製造出新的血液與免疫系統而延續病患生命。 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.
臍帶血中的造血幹細胞及前驅細胞的濃度比其在骨髓內多10-20倍,增生能力也較好。除此之外,其配對要求較低,非親屬間骨髓移植要求六個白血球抗原皆符合,臍帶血幹細胞移植則只要四或五個抗原相同即可。 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.
然而,可採集的臍帶血幹細胞數量有限,若使用於幼兒時期尚可,若使用於成人,則稍顯不足。過去研究顯示同位序列基因(homeobox gene)HOXB4對於造血幹細胞自我更新的調控十分重要,可維持它在骨髓中的群集大 小。 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.
加拿大蒙特婁大學〈University of Montreal〉的蓋˙薩瓦格(Guy Sauvageau)博士及其團隊針對骨髓中幹細胞數目受限的情況進行研究,其團隊是最早利用人類及老鼠的細胞株證明HOX基因會在血球細胞表現。其中HOXB4蛋白質已最常被用於作為活體外造血幹細胞(HSC)增生之有效刺激劑。 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.
為了提升HOXB4蛋白質用於造血幹細胞增生刺激劑之效果,已有數個研究致力於利用點突變或序列片段刪除技術以改進該蛋白質之穩定度。例如美國專利公告號8039436B2一案中揭示了將HOXB4胺基酸序列第6、7、23及28個胺基酸中至少一個取代為親脂性胺基酸,以及刪除胺基酸序列第31至35的胺基酸,都有助於降低該蛋白質經由泛素(ubiquitin)的標記而被蛋白酶體(proteasome)水解的影響。而美國專利公告號9783783B2一案則揭示了對HOXB4上的LEXE模體(motif)進行點突變(胺基酸序列第175、176及178個胺基酸),可有效降低CUL4所媒介的蛋白質水解。然而,目前尚未有研究揭露突變型HOXB4蛋白質具有更佳的去氧核醣核酸(DNA)結合能力。 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同時為一DNA結合蛋白質,在結構上包含有一DNA鍵結區域,先前已委託弘光科技大學陳曄博士藉由X-ray射線晶體技術解出HOXB4的DNA鍵結區域之結構(圖1)。其結構與蛋白質資料庫中名稱為1B72之HOXB1蛋白質結構(圖2)相似。根據HOXB4蛋白質的DNA鍵結區域與DNA鍵結之模擬三維結構圖,位於結合區域序列上第47、48與59個胺基酸在DNA鍵結扮演重要的角色(圖3)。這三個位置分別為精胺酸(Arginine,R)、麩醯胺酸(Glutamine,Q)與離胺酸(Lysine,K),其對應在HOXB4全長序列分別為第204、205及216個胺基酸,為求一致性,將以R204、Q205與K216使用於後續描述。 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.
於是,本發明藉由基因工程技術,針對HOXB4蛋白質的DNA結合區域之上述所提三個特定位置,進行胺基酸變異,以聚合酶連鎖反應進行 質體點突變,進而改變其蛋白質階段的胺基酸。經由本發明所製得之單一突變點重組蛋白質與雙突變點重組蛋白質與DNA結合能力皆有所提升,且雙突變點重組蛋白質之幹細胞增生能力優於野生型HOXB4蛋白質。 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.
基於以上之目的,本發明係針對HOXB4蛋白質的DNA結合區域之上述所提三個特定位置,進行胺基酸變異,以聚合酶連鎖反應進行質體點突變,以得到相較於野生型HOXB4蛋白質,具有與DNA分子更好的結合能力,以及更佳之幹細胞增生能力。 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.
於是,本發明之一方面係關於一種突變homeobox B4(HOXB4)蛋白質,其包含如下胺基酸序列:於序列編號2所表示之胺基酸序列或與其具有至少95%之一致性之胺基酸序列中,相當於序列編號2所表示之胺基酸序列之第204、205及216位置的至少一位置之胺基酸係經變異者,且與包含該序列編號2所表示之胺基酸序列或與其具有至少95%之一致性之胺基酸序列的野生型HOXB4蛋白質相比,該突變HOXB4蛋白質具有更高之與DNA之結合能力。 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.
於本發明之一些具體實施態樣,上述之蛋白質,其中相當於序列編號2所表示之胺基酸序列之第204、205及216位中至少一位置之胺基酸係經變異為中性或帶正電之胺基酸。 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.
於本發明之一些具體實施態樣,上述之HOXB4蛋白質,其中至少包含一種以下之變異:相當於序列編號2所表示之胺基酸序列之第204位置之胺基酸係經變異為組胺酸(Histidine;His;H)或離胺酸(Lysine;Lys;K);相當於序列編號2所表示之胺基酸序列之第205位置之胺基酸係經變異為組胺酸(H)或精胺酸(Arginine;Arg;R);及相當於序列編號2所表示之胺基酸序列之第216位置之胺基酸係經變異 為組胺酸(H)或精胺酸(R)。 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).
於本發明之其他具體實施態樣,上述之HOXB4蛋白質,其中相當於序列編號2所表示之胺基酸序列之第205及216位置之胺基酸係分別經變異為組胺酸(H)及精胺酸(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).
本發明之又一方面,係關於一種重組蛋白質包含上述之HOXB4蛋白質及一細胞穿透胜肽(cell-penetrating peptide)。 Another aspect of the present invention relates to a recombinant protein comprising the above-mentioned HOXB4 protein and a cell-penetrating peptide.
於本發明之一些具體實施態樣,所述之重組蛋白質,其中該細胞穿透胜肽係一轉錄活化因子(trans-activator of transcription,TAT)。 In some embodiments of the present invention, the recombinant protein, wherein the cell-penetrating peptide is a trans-activator of transcription (TAT).
於本發明之其他具體實施態樣,所述之重組蛋白質,其中該轉錄活化因子係包含序列編號4之序列。 In other specific embodiments of the present invention, the recombinant protein, wherein the transcription activator comprises the sequence of SEQ ID NO: 4.
本發明之又一方面,係關於一種細胞擴增之方法,包含將促進細胞擴增之有效量之請求項1之HOXB4蛋白質投與至目標細胞,以使細胞擴增。
Yet another aspect of the present invention relates to a method for cell expansion, comprising administering an effective amount of the HOXB4 protein of
於本發明之一些具體實施態樣,上述之方法,係在體外將促進細胞擴增之有效量之HOXB4蛋白質投與至目標細胞。 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.
於本發明之一些具體實施態樣,上述之方法,其中該目標細胞係前驅細胞造血幹細胞、及/或由該等分化誘導而成之細胞。 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.
於本發明之一些具體實施態樣,上述之方法,其中該目標細胞係一造血幹細胞。 In some embodiments of the present invention, the above method, wherein the target cell is a hematopoietic stem cell.
於本發明之一些具體實施態樣,上述之方法,其中該造血幹細胞之來源為臍帶血。 In some embodiments of the present invention, in the above method, the source of the hematopoietic stem cells is umbilical cord blood.
於本發明之一些具體實施態樣,上述之方法,其中該造血幹細胞之來源為週邊血。 In some embodiments of the present invention, in the above method, the source of the hematopoietic stem cells is peripheral blood.
於本發明之一些具體實施態樣,上述之方法,其中該造血幹細胞之來源為骨髓。 In some embodiments of the present invention, in the above method, the source of the hematopoietic stem cells is bone marrow.
圖1 X-ray射線晶體技術解出之HOXB4蛋白質的DNA鍵結區域之結構。 Fig. 1 The structure of the DNA-binding region of HOXB4 protein solved by X-ray crystallography.
圖2 HOXB1蛋白質與Pbx1及DNA鍵結之結構圖(蛋白質資料庫Protein data bank ID:1B72) Figure 2. Structure diagram of HOXB1 protein binding to Pbx1 and DNA (Protein data bank ID: 1B72)
圖3 HOXB4蛋白質的DNA鍵結區域與DNA鍵結之模擬三維結構圖。鍵結區域序列上的第47位置精胺酸Arginine(R)、48麩醯胺酸Glutamine(Q)與第59位置離胺酸Lysine(K)與DNA鍵結扮演重要角色。 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.
圖4 為TAT-HOXB4野生型重組蛋白質胺基酸序列,其中以底線標示HOXB4全長序列,粗體標示DNA鍵結區域,並以反白標示欲進行變異之胺基酸位置。 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.
圖5 為pTAT-HA-HOXB4質體之目標DNA序列,其中以粗體標示DNA鍵結區域,並以反白標示欲進行變異之編碼序列位置。 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.
圖6 野生型/突變型TAT-HOXB4重組蛋白質可於大腸桿菌表現系統經由IPTG誘導產製,箭頭所指為目標蛋白質電泳位置。 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.
圖7 經純化之突變型TAT-HOXB4重組蛋白質。 Figure 7 Purified mutant TAT-HOXB4 recombinant protein.
圖8 列示以表面電漿共振生物感測器量測野生型與突變型TAT-HOXB4蛋白質與DNA結合情形。 Figure 8 shows the measurement of the binding of wild-type and mutant TAT-HOXB4 proteins to DNA by surface plasmon resonance biosensors.
圖9 列示以野生型與突變型TAT-HOXB4體外增生造血幹細胞之比較結果,以其CD34+細胞數量之測量值表示。對照組係以牛血清蛋白(Bovine Serum Albumin,BSA)處理。 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).
「幹細胞」是一種多能或多潛能細胞,具有自我更新及分化成不同種類細胞的能力。幹細胞自然存在於動物體內,藉由分化的潛能,提供動物所需之各種細胞來源。當幹細胞分裂,其子代細胞仍具有幹細胞特性,亦或者走向分化下游的路徑,成為成熟的成體細胞。 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.
「造血」細胞是指涉及造血過程(亦即前驅細胞形成成熟血液細胞之過程)的一種細胞。在成人,造血發生在骨髓。在發育早期的不同發育階段,造血發生在不同的地點;原始血液細胞出現於卵黃囊,後來,血液細胞在肝、脾和骨髓形成。造血是經歷複雜調控的過程,包括激素(例如紅血球生成素),生長因子(例如集落刺激因子)和細胞因子(例如介白素)。 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).
本發明係針對HOXB4的DNA鍵結區域之上述所提三個特定位置,進行胺基酸變異,其主要是藉由基因工程技術,以聚合酶連鎖反應進行質體點突變,進而改變其蛋白質階段的胺基酸。 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.
下列實施例係用以說明本發明之技術內容即可達成之功效,並非意欲用以限制本發明。凡依本發明所作之任何均等變化及修飾,在發明的範圍及精神內,皆屬本發明申請範圍之範疇。 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.
實施例1 點突變質體構築Example 1 Construction of point mutant plastids
使用pTAT-HA-HOXB4質體作為模板(如圖5),利用聚合酶連鎖反應(Polymerase Chain Reaction,PCR)針對特定位置R204、Q205與K216進行site-directed mutagenesis(定點突變)擴 增反應。以下列設計引子(如表一)分別各進行兩種胺基酸突變:Histidine(H)/Lysine(K)或H/Arginine(R),經由選殖與定序確認後,製得之新質體分別命名為pTAT-HA-HOXB4_R204H、pTAT-HA-HOXB4_R204K、pTAT-HA-HOXB4_Q205H、pTAT-HA-HOXB4_Q205R、pTAT-HA-HOXB4_K216H及pTAT-HA-HOXB4_K216R。雙點突變則以pTAT-HA-HOXB4_Q205H作為模板,利用表一之K216R引子組進行定點突變擴增反應,新質體命名為pTAT-HA-HOXB4_Q205H_K216R。 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.
實施例2 野生型與點突變型TAT-HOXB4重組蛋白質之表現純化比較Example 2 Comparison of expression and purification of wild-type and point mutant TAT-HOXB4 recombinant proteins
將(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分別轉型至大腸桿菌菌株BL21(DE3)pLysS(Novagen)。經轉型後之大腸桿菌細胞於37℃以150rpm旋轉震盪培養過夜。將過夜培養液以1:50稀釋至新鮮培養液,起始OD600值約為0.05,並於37℃以200rpm旋轉震盪生長,直至 OD600值為0.7-0.8,接著以1mM異丙基硫代-b-D-半乳糖(IPTG)於37℃以200rpm旋轉震盪進行誘導蛋白質表現3小時。 (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.
誘導作用後,將細胞離心收集,並以緩衝液A(8M尿素,20mM HEPES,100mM NaCl,pH 8.0)回溶菌體。將此細胞懸浮液以高壓差均質機French Press擠壓三次,並將細胞裂解產物以18,000rpm於4℃離心30分鐘使其澄清,並收取上清液。將上清液調整至含有10mM咪唑(imidazole)之緩衝液,並將樣品加至HisTrap親和性管柱(GE Healthcare),將結合之蛋白質以含有50、100、150、300、500及1000mM咪唑之緩衝液A進行溶析。收集含有突變型HOXB4之溶析樣品,加至HiTrap SP陽離子交換層析管柱,並以不含咪唑與尿素之緩衝液B(20mM HEPES,1M NaCl,pH 8.0)利用梯度進行溶析。收集含有目標蛋白質突變型HOXB4之溶析樣品,並以透析方式進行蛋白質再摺疊。將再摺疊蛋白質樣品分裝,並以液態氮急速冷凍後,保存於-80℃。 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.
經由點突變後之質體所表現之蛋白質,其理論分子量大小及等電點與野生型HOXB4(HOXB4_WT)差異極微小,如表2。 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.
實施例3 野生型與點突變型TAT-HOXB4重組蛋白質與去氧核醣核酸DNA結合的能力Example 3 The ability of wild-type and point mutant TAT-HOXB4 recombinant proteins to bind to deoxyribonucleic acid DNA
利用Surface plasmon resonance(SPR)表面電漿共振生物感測器量測HOXB4蛋白質與DNA HOXB4 binding consensus DNA sequence(結合共有DNA序列:5’-CTGCGATGATTGATGACCGC-3’。)間的動態結合與解離過程,比較野生型HOXB4(序列編號32)與突變型HOXB4(包含單一突變R204H(序列編號34)、R204K(序列編號36)、Q205H(序列編號38)、Q205R(序列編號40)、K216H(序列編號42)、K216R(序列編號44)以及雙點突變Q205H_K216R(序列編號46))與DNA結合的能力。由圖8的數據可知,除了HOXB4_K216H的結合能力與野生型HOXB4相似外,其他單一突變之HOXB4重組蛋白質與DNA結合能力皆有1.39-1.89倍的增強現象,而雙點突變重組蛋白質HOXB4_Q205H_K216R與DNA之結合能力為野生型HOXB4的50倍。以此方法量測之重組蛋白質與DNA之結合能力,單一突變型以HOXB4_R204K最強,HOXB4_Q205R次之。雙點突變重組蛋白質HOXB4_Q205H_K216R與DNA結合能力遠優於任一單一點突變重組蛋白質以及野生型HOXB4。 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.
實施例4 野生型與點突變型TAT-HOXB4重組蛋白質對於體外造血幹細胞增生的能力Example 4 The ability of wild-type and point mutant TAT-HOXB4 recombinant proteins to proliferate hematopoietic stem cells in vitro
將人類臍帶血中之紅血球藉由於4℃下於含有0.1% 碳酸氫鈉之0.83%氯化銨(pH 7.0)中進行溶解而去除。將經收集之白血球部分與磁珠結合之特定抗體與細胞共同培養後,藉由Stemsep管柱分離方式進行磁性細胞分離程序以收集帶CD34+細胞,並除去表現成熟人類白血球細胞之部分。取特定密度之細胞於六孔盤培養,並於早晚加入特定濃度之BSA或野生型或突變型TAT-HOXB4重組蛋白質,連續進行4天,於第5天以flow(LIN/CD34染色加正向與側向散射光)計數幹細胞數量並進行分析。 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.
由圖9之結果證明,根據本發明所製得之單一點突變TAT-HOXB4重組蛋白質對於臍帶血幹細胞之增生能力,以TAT-HOXB4_Q205H最佳,與野生型TAT-HOXB4相當,TAT-HOXB4_K216R次之。而根據此結果更進一步進行雙點突變之重組蛋白質TAT-HOXB4_Q205H_K216R對於造血幹細胞優於前述兩者,且略優於野生型TAT-HOXB4。 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.
綜合上述,由本案實施例之DNA鍵結能力及幹細胞增生實驗數據顯示,經由本發明所製得之單一突變點重組蛋白質與DNA結合能力提升1.4~1.9倍,且HOXB4_Q205H具有與野生型HOXB4重組蛋白質相當的幹細胞增生能力。而根據單一突變點重組蛋白質之幹細胞增生能力分析數據,選擇於Q205與K216兩位置進行雙點胺基酸突變HOXB4_Q205H_K216R,此雙點突變重組蛋白質之DNA結合能力與野生型HOXB4相比提升50倍。經本發明方法製得之突變型雙點突變重組蛋白質HOXB4_Q205H_K216R重組蛋白質,同樣可用於幹細胞增生,且幹細胞增生能力優於野生型HOXB4。 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.
<110> 台灣尖端先進生技醫藥股份有限公司 <110> Taiwan Advanced Biotech Co., Ltd.
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<213> 人造序列 <213> Artificial Sequences
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<223> HOXB4-Q205R <223> HOXB4-Q205R
<400> 24 <400> 24
<210> 25 <210> 25
<211> 756 <211> 756
<212> DNA <212> DNA
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> HOXB4-K216H <223> HOXB4-K216H
<400> 25 <400> 25
<210> 26 <210> 26
<211> 251 <211> 251
<212> PRT <212> PRT
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> HOXB4-K216H <223> HOXB4-K216H
<400> 26 <400> 26
<210> 27 <210> 27
<211> 756 <211> 756
<212> DNA <212> DNA
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> HOXB4-K216R <223> HOXB4-K216R
<400> 27 <400> 27
<210> 28 <210> 28
<211> 251 <211> 251
<212> PRT <212> PRT
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> HOXB4-K216R <223> HOXB4-K216R
<400> 28 <400> 28
<210> 29 <210> 29
<211> 756 <211> 756
<212> DNA <212> DNA
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> HOXB4-Q205H-K216R <223> HOXB4-Q205H-K216R
<400> 29 <400> 29
<210> 30 <210> 30
<211> 251 <211> 251
<212> PRT <212> PRT
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> HOXB4-Q205H-K216R <223> HOXB4-Q205H-K216R
<400> 30 <400> 30
<210> 31 <210> 31
<211> 960 <211> 960
<212> DNA <212> DNA
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4 <223> TAT-HOXB4
<400> 31 <400> 31
<210> 32 <210> 32
<211> 319 <211> 319
<212> PRT <212> PRT
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4 <223> TAT-HOXB4
<400> 32 <400> 32
<210> 33 <210> 33
<211> 960 <211> 960
<212> DNA <212> DNA
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_R204H <223> TAT-HOXB4_R204H
<400> 33 <400> 33
<210> 34 <210> 34
<211> 319 <211> 319
<212> PRT <212> PRT
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_R204H <223> TAT-HOXB4_R204H
<400> 34 <400> 34
<210> 35 <210> 35
<211> 960 <211> 960
<212> DNA <212> DNA
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_R204K <223> TAT-HOXB4_R204K
<400> 35 <400> 35
<210> 36 <210> 36
<211> 319 <211> 319
<212> PRT <212> PRT
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_R204K <223> TAT-HOXB4_R204K
<400> 36 <400> 36
<210> 37 <210> 37
<211> 960 <211> 960
<212> DNA <212> DNA
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_Q205H <223> TAT-HOXB4_Q205H
<400> 37 <400> 37
<210> 38 <210> 38
<211> 319 <211> 319
<212> PRT <212> PRT
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_Q205H <223> TAT-HOXB4_Q205H
<400> 38 <400> 38
<210> 39 <210> 39
<211> 960 <211> 960
<212> DNA <212> DNA
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_Q205R <223> TAT-HOXB4_Q205R
<400> 39 <400> 39
<210> 40 <210> 40
<211> 319 <211> 319
<212> PRT <212> PRT
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_Q205R <223> TAT-HOXB4_Q205R
<400> 40 <400> 40
<210> 41 <210> 41
<211> 960 <211> 960
<212> DNA <212> DNA
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_K216H <223> TAT-HOXB4_K216H
<400> 41 <400> 41
<210> 42 <210> 42
<211> 319 <211> 319
<212> PRT <212> PRT
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_K216H <223> TAT-HOXB4_K216H
<400> 42 <400> 42
<210> 43 <210> 43
<211> 960 <211> 960
<212> DNA <212> DNA
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_K216R <223> TAT-HOXB4_K216R
<400> 43 <400> 43
<210> 44 <210> 44
<211> 319 <211> 319
<212> PRT <212> PRT
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_K216R <223> TAT-HOXB4_K216R
<400> 44 <400> 44
<210> 45 <210> 45
<211> 960 <211> 960
<212> DNA <212> DNA
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_Q205H_K216R <223> TAT-HOXB4_Q205H_K216R
<400> 45 <400> 45
<210> 46 <210> 46
<211> 319 <211> 319
<212> PRT <212> PRT
<213> 人造序列 <213> Artificial Sequences
<220> <220>
<223> TAT-HOXB4_Q205H_K216R <223> TAT-HOXB4_Q205H_K216R
<400> 46 <400> 46
Claims (11)
Priority Applications (2)
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TW108106827A TWI756518B (en) | 2019-02-27 | 2019-02-27 | Mutated recombinant hoxb4 proteins, method for manufacturing same, and use thereof |
CN201910421421.4A CN111620938B (en) | 2019-02-27 | 2019-05-21 | Mutant HOXB4 recombinant protein and preparation method and application thereof |
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Citations (2)
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CN101016540A (en) * | 2006-02-10 | 2007-08-15 | 台湾尖端先进生技医药股份有限公司 | Method of preparing HOXB4 recombination protein with histidine mark at C-end and use of the same |
US8039436B2 (en) * | 2007-06-01 | 2011-10-18 | British Columbia Caner Agency Branch | Mutated HOXB4 proteins with improved stability, and methods of use thereof |
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US8222206B2 (en) * | 2008-03-04 | 2012-07-17 | Taiwan Advance Bio-Pharm Inc. | Method of enhancing the mobilization of hematopoietic stem cells using TAT-HOXB4H |
WO2014120714A1 (en) * | 2013-01-30 | 2014-08-07 | Cornell University | Compositions and methods for the expansion of stem cells |
-
2019
- 2019-02-27 TW TW108106827A patent/TWI756518B/en active
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CN101016540A (en) * | 2006-02-10 | 2007-08-15 | 台湾尖端先进生技医药股份有限公司 | Method of preparing HOXB4 recombination protein with histidine mark at C-end and use of the same |
US8039436B2 (en) * | 2007-06-01 | 2011-10-18 | British Columbia Caner Agency Branch | Mutated HOXB4 proteins with improved stability, and methods of use thereof |
Non-Patent Citations (1)
Title |
---|
Jana Krosl.ET AL. "In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4 protein" Nature Medicine 9(2003/10/26):1428–1432 * |
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