人肿瘤抑制因子Folliculin(FLCN)蛋白的原核表达纯化
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摘要
肿瘤抑制因子Folliculin(FLCN)的突变或缺失会引起常染色体显性遗传病——BHD综合征,但其诱发机制不清,因而认知FLCN的结构和功能非常必要。本文利用优化pGEX-6p-1-GST-FLCN重组质粒构建了大肠杆菌原核表达系统,诱导表达GST-FLCN融合蛋白,再通过GST介质亲和层析和分子筛纯化,最后通过Xa因子蛋白酶切除GST标签,收集获取了高纯度(90%左右)的FLCN蛋白,为FLCN的结构与功能分子奠定了基础。
Folliculin(FLCN)is a tumor suppressor,its mutation or deletion causes autosomal dominant genetic disease BHD syndrome,but its induction mechanism is unclear.Therefore,it is necessary to recognize the structure and function of FLCN.In this paper,the prokaryotic expression system of E.coli was constructed by optimizing the recombinant plasmid pGEX-6p-1-GST-FLCN,which induced the expression of GST-FLCN fusion protein,and then purified by GST medium affinity chromatography and molecular sieve.Finally,the GST label was excised by factor Xa protease.The collection of high purity FLCN protein laid the foundation for the structural and functional molecules of FLCN.
Folliculin(FLCN)is a tumor suppressor,its mutation or deletion causes autosomal dominant genetic disease BHD syndrome,but its induction mechanism is unclear.Therefore,it is necessary to recognize the structure and function of FLCN.In this paper,the prokaryotic expression system of E.coli was constructed by optimizing the recombinant plasmid pGEX-6p-1-GST-FLCN,which induced the expression of GST-FLCN fusion protein,and then purified by GST medium affinity chromatography and molecular sieve.Finally,the GST label was excised by factor Xa protease.The collection of high purity FLCN protein laid the foundation for the structural and functional molecules of FLCN.
引文
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[7]Vocke CD,Yang Y,Pavlovich CP,et al.High Frequency of Somatic Frameshift BHD Gene Mutations in Birt-Hogg-DubeAssociated Renal Tumors[J].Journal of the National Cancer Institute,2005,97(12):931-935.
[8]Singh SR,Zhen W,Zheng Z,et al.The Drosophila Homolog of the Human Tumor Suppressor Gene BHD Interacts with the JAK-STATand Dpp Signaling Pathways in Regulating Male Germline Stem Cell Maintenance[J].Oncogene,2006,25(44):5933-5941.
[9]Nookala RK,Langemeyer L,Pacitto A,et al.Crystal Structure of Folliculin Reveals a hidDENN Function in Genetically Inherited Renal Cancer[J].Open biology,2012,2(8):120071.
[10]Hasumi H,Hasumi Y,Baba M,et al.H255Y and K508R Missense Mutations in Tumour Suppressor Folliculin(FLCN)Promote Kidney Cell Proliferation[J].Human molecular genetics,2017,26(2):354-566.
[11]Goncharova EA,Goncharov DA,James ML,et al.Folliculin Controls Lung Alveolar Enlargement and Epithelial Cell Survival Through E-Cadherin,LKB1,and AMPK[J].Cell reports,2014,7(2):412-423.
[2]Scalvenzi M,Argenziano G,Sammarco E,et al.Hereditary Multiple Fibrofolliculomas,Trichodiscomas and Acrochordons:Syndrome of Birt-Hogg-Dube[J].Journal of the European Academy of Dermatology and Venereology:JEADV,1998,11(1):45-47.
[3]Toro JR,Glenn G,Duray P,et al.Birt-Hogg-Dube Syndrome:a Novel Marker of Kidney Neoplasia[J].Archives of dermatology,1999,135(10):1195-1202.
[4]Khoo SK,Bradley M,Wong FK,et al.Birt-Hogg-Dube Syndrome:Mapping of a Novel Hereditary Neoplasia Gene to Chromosome17p12-q11.2[J].Oncogene,2001,20(37):5239-5242.
[5]Nickerson ML,Warren MB,Toro JR,et al.Mutations in a Novel Gene Lead to Kidney Tumors,Lung Wall Defects,and Benign Tumors of the Hair Follicle in Patients with the Birt-Hogg-Dube Syndrome[J].Cancer cell,2002,2(2):157-164.
[6]Yang Y,Padilla-Nash HM,Vira MA,et al.The UOK 257 Cell Line:a Novel Model for Studies of the Human Birt-Hogg-Dube Gene Pathway[J].Cancer Genetics and Cytogenetics,2008,180(2):100-109.
[7]Vocke CD,Yang Y,Pavlovich CP,et al.High Frequency of Somatic Frameshift BHD Gene Mutations in Birt-Hogg-DubeAssociated Renal Tumors[J].Journal of the National Cancer Institute,2005,97(12):931-935.
[8]Singh SR,Zhen W,Zheng Z,et al.The Drosophila Homolog of the Human Tumor Suppressor Gene BHD Interacts with the JAK-STATand Dpp Signaling Pathways in Regulating Male Germline Stem Cell Maintenance[J].Oncogene,2006,25(44):5933-5941.
[9]Nookala RK,Langemeyer L,Pacitto A,et al.Crystal Structure of Folliculin Reveals a hidDENN Function in Genetically Inherited Renal Cancer[J].Open biology,2012,2(8):120071.
[10]Hasumi H,Hasumi Y,Baba M,et al.H255Y and K508R Missense Mutations in Tumour Suppressor Folliculin(FLCN)Promote Kidney Cell Proliferation[J].Human molecular genetics,2017,26(2):354-566.
[11]Goncharova EA,Goncharov DA,James ML,et al.Folliculin Controls Lung Alveolar Enlargement and Epithelial Cell Survival Through E-Cadherin,LKB1,and AMPK[J].Cell reports,2014,7(2):412-423.