人可溶性CD105蛋白的真核表达及纯化
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摘要
目的建立一种简便、高效的人可溶性CD105(soluble CD105,sCD105)蛋白的真核表达及纯化方法。方法人工合成胞外区和信号肽区域的DNA片段,在其信号肽前端加入Kozak sequence(GCACCATGG)序列,促进蛋白表达,同时在其C-末端6×His前加入K(AAG)氨基酸,促进6×His的暴露有助于后期纯化,构建真核表达质粒pcDNA3. 4-sCD105。通过瞬时转染的方法将重组质粒转染293F悬浮细胞,收集细胞上清液,利用His-trap EXCEL柱通过两步纯化法进行纯化。将纯化获得的sCD105蛋白超滤浓缩后,进行10%SDS-PAGE、Western blot及ELISA检测。结果在20 mL(1×10~6个/mL)293F悬浮细胞中表达及纯化后,获得纯度> 95%的sCD105蛋白3 mL,浓度为27. 83 mg/L,获得率高达4. 17 mg/L。结论成功建立了一种简便高效的sCD105蛋白表达及纯化的方法,为后期蛋白功能的研究及相应治疗或诊断性抗体的开发奠定了基础。
Objective To develop a simple and effective method for eukaryotic expression and purification of human soluble CD105(sCD105) protein. Methods The DNA fragments of extracellular domain and signal peptide domain of sCD105 were synthesized. A Kozak sequence(GCACCATGG)was added in front of signal peptide to promote the protein expression,while K(AAG) in front of 6 × His at C-terminus to promote the exposure of 6 × His and the protein purification,based on which a eukaryotic expression vector pcDNA3. 4-sCD105 was constructed and transfected to 293 F suspension cells. The supernatant of transfected cells was collected and purified by two-step HIS-trap EXCEL column chromatography. The obtained sCD105 protein was concentrated by ultrafiltration,and analyzed by SDS-PAGE,Western blot and ELISA. Results A portion of 3 mL of sCD105 protein with a purity of more than 95% was isolated by the developed method from 20 mL(1 × 10~6 cells/mL)of 293 F cells,of which the concentration was 27. 83 mg/L and the yield was 4. 17 mg/L. Conclusion A simple and effective method for expression and purification of sCD105 protein was developed,which laid a foundation of study on the function of sCD105 protein and development of the corresponding therapeutic and diagnostic antibodies.
Objective To develop a simple and effective method for eukaryotic expression and purification of human soluble CD105(sCD105) protein. Methods The DNA fragments of extracellular domain and signal peptide domain of sCD105 were synthesized. A Kozak sequence(GCACCATGG)was added in front of signal peptide to promote the protein expression,while K(AAG) in front of 6 × His at C-terminus to promote the exposure of 6 × His and the protein purification,based on which a eukaryotic expression vector pcDNA3. 4-sCD105 was constructed and transfected to 293 F suspension cells. The supernatant of transfected cells was collected and purified by two-step HIS-trap EXCEL column chromatography. The obtained sCD105 protein was concentrated by ultrafiltration,and analyzed by SDS-PAGE,Western blot and ELISA. Results A portion of 3 mL of sCD105 protein with a purity of more than 95% was isolated by the developed method from 20 mL(1 × 10~6 cells/mL)of 293 F cells,of which the concentration was 27. 83 mg/L and the yield was 4. 17 mg/L. Conclusion A simple and effective method for expression and purification of sCD105 protein was developed,which laid a foundation of study on the function of sCD105 protein and development of the corresponding therapeutic and diagnostic antibodies.
引文
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[2] GOUGOS A,LETARTE M. Primary structure of endoglin,an RGD-containing glycoprotein of human endothelial cells[J]. J Biol Chem,1990,265(15):8361-8364.
[3] OLLAURIIB魣譙EZ C,L仵PEZNOVOA J M,PERICACHO M.Endoglin-based biological therapy in the treatment of angiogenesis-dependent pathologies[J]. Expert Opin Biol Ther,2017,17(9):1-11.
[4] ROSSI E,PERICACHO M,BACHELOT-LOZA C,et al. Human endoglin as a potential new partner involved in plateletendothelium interactions[J]. Cell Mol Life Sci,2017,75(7):1269-1284.
[5] PLUMITALLO S,RUIZLLORENTE L,LANGA C,et al. Functional analysis of a novel ENG variant in a patient with hereditary hemorrhagic telangiectasia(HHT)identifies a new Sp1binding-site[J]. Gene,2018,647:85-92.
[6] SUGDEN W W,SIEKMANN A F. Endothelial cell biology of Endoglin in hereditary hemorrhagic telangiectasia[J]. Curr Opin Hematol,2018,25(3):1.
[7] BOURDEAU A,DUMONT D J,LETARTE M. A murine model of hereditary hemorrhagic telangiectasia[J]. J Clin Investig,1999,104(10):1343-1351.
[8] RUIZREMOLINA L,OLLAURILBEZ C,P魪REZROQUE L,et al. Circulating soluble endoglin modifies the inflammatory response in mice[J]. Plos One,2017,12(11):e0188204.
[9] KOKAJI E,SHIMOMURA A,MINAMISAKA T,et al. Endoglin(CD105)and SMAD4 regulate spheroid formation and the suppression of the invasive ability of human pancreatic cancer cells[J]. Int J Oncol,2018,52(3):892-900.
[10] VAREJCKOVA M,GALLARDO-VARA E,VICEN M,et al.Soluble endoglin modulates the pro-inflammatory mediators NF-κB and IL-6 in cultured human endothelial cells[J]. Life Sci,2017,175:52-60.
[11] LEBRIN F,GOUMANS M J,JONKER L,et al. Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction[J]. Embo J,2014,23(20):4018-4028.
[12] TIAN H Y,KETOVA T,HARDY D,et al. Endoglin mediates vascular maturation by promoting vascular smooth muscle cell migration and spreading[J]. Arterioscler Thromb Vascular Biol,2017,37(6):1115-1126.
[13] PAAUWE M,HEIJKANTS R C,OUDT C H,et al. Endoglin targeting inhibits tumor angiogenesis and metastatic spread in breast cancer[J]. Oncogene,2016,35(31):4069-4079.
[14] Structural and functional characterization of soluble endoglin receptor[J]. Biochem&Biophys Res Commun,2009,383(4):386-391.