DEPTOR蛋白的表达纯化条件及与mTOR-TRD2亚结构域的相互作用

doi:10.7503/cjcu20150817

摘要/Abstract

摘要：

测试并优化了哺乳动物雷帕霉素靶蛋白(mTOR)的FAT结构域及其亚结构域TRD2和DEPTOR蛋白在大肠杆菌中的表达条件, 获得了TRD2和DEPTOR蛋白成功表达并大量纯化的条件. 结果表明, 麦芽糖结合蛋白(MBP)融合mTOR的TRD2亚结构域可以在菌株BL21(DE3)中大量表达. DEPTOR-G270P突变比野生型DEPTOR在BL21(DE3)中的表达量提高约5倍, 且该突变并不影响DEPTOR自身的二级结构. 通过凝胶过滤实验发现, mTOR的TRD2亚结构域和DEPTOR之间可能存在相互作用.

关键词: mTOR蛋白, TRD2亚结构域, DEPTOR蛋白, G270P突变, 大肠杆菌表达

Abstract:

The inhibition of mammalian target of rapamycin(mTOR) is important to cancer treatment. DEPTOR is currently known as an endogenous mTOR inhibitor and could interact with mTOR FAT domain to suppress mTOR activity in vivo. It showed that MBP fusion mTOR TRD2 subdomain could be expressed in BL21(DE3) with conversional protocol and DEPTOR-G270P mutation could be expressed 5-fold more than wild-type DEPTOR in BL21(DE3). Adding glycerol(volume fraction 10%) in purification buffer would improve DEPTOR-G270P purity. CD spectra assay showed that this purification protocol could produce DEPTOR in large quantity without affecting its secondary structure. Gel-filtration assay indicated possible interactions between mTOR-TRD2 subdomain and DEPTOR, which may provide insights into mTOR-DEPTOR interaction and give new information about mTOR inhibition.

Key words: Mammalian target of rapamycin(mTOR), TRD2 subdomain, DEPTOR, G270P mutation, Escherichia coli expression

中图分类号:

O629
Q516

TrendMD:

王男, 程小桁, 郑积敏, 陈光巨, 贾宗超. DEPTOR蛋白的表达纯化条件及与mTOR-TRD2亚结构域的相互作用. 高等学校化学学报, 2016, 37(5): 912.

WANG Nan, CHENG Xiaoheng, ZHENG Jimin, CHEN Guangju, JIA Zongchao. Expression and Purification of DEPTOR and Its Interaction with mTOR-TRD2^†. Chem. J. Chinese Universities, 2016, 37(5): 912.

图/表 9

Table 1 FAT domain and TRD2 subdomain PCR primer sequence

Primer name	Sequence(5' to 3')
FAT foward primer	CTGCTGGGTGAGAGAGCT
FAT reverse primer	AGACTTAGAAGCCACTGTCAGT
TRD2 forward primer	ATCCAGGCTACCTGGTATGA
TRD2 reverse primer	CTTGTCCCCGAGCGAC

Table 2 DEPTOR PCR Primer and G270P Mutation Primer Sequence

Primer name	Sequence(5' to 3')
DEPTOR foward primer	GCTTGGTACCGAGCTCGG
DEPTOR reverse primer	ATGGTGATGGTGATGATG
DEPTOR-G270P-forward	GGCAGCAGCCCCTACTTCAGCAGCAGCCCCACCC
DEPTOR-G270P-reverse	TGAAGTAGGGGCTGCTGCCACAGCTGCTCATGCTGCTTCT

Fig.1 Sub-domains of mTOR FAT domainThe 0—600 number suggests amino acids numbers in primary structure.

Fig.2 FAT domain and subdomain expression assay in E.coli(A) FAT-GST fusion protein in JM109 expression assay by Western-Blot, lane 1: negative control (incubate without adding IPTG), lane 2: whole-cell-extract after induced by 1 mmol/L IPTG, lanes 3, 4: supernatant/pellet of whole-cell-extract lysate. (B) MBP fusion TRD2 protein in BL21(DE3) expression assay by SDS-PAGE(Ft: flow-through; W: wash; E: eluted protein).

Fig.3 DEPTOR expression assay in E.coliAll the lanes are samples from each purification step, respectively. (A) DEPTOR-His wild-type expression assay by Western-Blot in BL21(DE3); (B) DEPTOR-His wild-type expression assay by Western-Blot in arctic express; (C) DEPTOR-His wild-type expression assay by SDS-PAGE in BL21(DE3); (D) DEPTOR-His G270P expression assay by SDS-PAGE; (E) DEPTOR-His G270P expression assay by SDS-PAGE with glycerol(10%, volume fraction) in purification buffer. P: pellet; S: supernatant; Cell: whole-cell-extract; Control: cell without induced by IPTG; Ft: flow-through; W1, W2: wash; E, E1 and E2: eluted protein; R: resin after elution.

Table 3 Percentage of secondary structure in DEPTOR-wt*

Secondary structure	Percentage of secondary structure(%)
Secondary structure	180—260 nm	185—260 nm	190—260 nm	195—260 nm	200—260 nm	205—260 nm
α-Helix	29.1	28.8	28.8	27.8	26.7	27.6
Antiparallel β-strand	14.2	14.6	15.0	13.4	11.0	9.4
Parallel β-strand	9.1	9.0	8.9	9.8	10.5	10.9
β-Turns	18.3	18.3	18.2	18.1	18.4	17.8
Coiled-coil	31.8	32.2	31.8	33.6	35.9	37.7
Total	102.4	102.9	102.8	102.7	102.5	103.3

Table 4 Percentage of secondary structure in DEPTOR-G270P*

Secondary structure	Percentage of secondary structure(%)
Secondary structure	180—260 nm	185—260 nm	190—260 nm	195—260 nm	200—260 nm	205—260 nm
α-Helix	28.8	29.4	29.3	28.6	28.0	29.4
Antiparallel β-strand	14.9	14.2	14.7	12.9	10.5	8.8
Parallel β-strand	9.1	8.8	8.7	9.5	10.0	10.3
β-Turns	18.4	18.2	18.1	17.9	18.1	17.4
Coiled-coil	31.0	31.2	31.0	32.6	34.7	36.2
Total	102.2	101.8	101.9	101.6	101.3	102.1

Fig.4 Location of TRD2 subdomain in mTOR-FAT domain(circle)

Fig.5 Interaction assay of MBP-TRD2 and DEPTOR(A) MBP-TRD2 and DEPTOR co-purification by gel-filtration (Hi-Load S200) in 20 mmol/L Tris, pH=8.0 and 150 mmol/L NaCl; (B) MBP and DEPTOR co-purification by gel-filtration in the same condition as a negative control. Samples of the putative complexes are picked and analyzed by SDS-PAGE. In each figure, x-axis indicates the elution volume which reflects molecular mass of the protein and y-axis indicates the absorption value which is in direct proportion to protein concentration.

参考文献 23

[1]	Wang J. B., Jiang Y., Liang H., Li P., Xiao H. J., Ji J., Xiang W., Shi J. F., Fan Y. G., Li L., Wang D., Deng S. S., Chen W. Q., Wei W. Q., Qiao Y. L., Boffetta P., Ann.Oncol., 2001, 23(11), 2983—2989
[2]	Qiao Y. L., Chin. J.Oncol., 2009, 34(7), 483—485
[3]	Wei K. R., Liang Z. H., Liu J., Wang Y. N., Chinese Journal of Medical History, 2012, 42(1), 21—25
	(魏矿荣, 梁智恒, 刘静, 王亚娜. 中华医史杂志, 2012, 42(1), 21—25)
[4]	Fan W. H., Zhang L. H., Zhang Z. L., Guo J. F., Ren A. M., Ge P. F., Chem. J. Chinese Universities,2013, 34(7), 1731—1738
	(范文海, 张丽红, 张子龙, 郭景富, 任爱民, 葛鹏飞. 高等学校化学学报, 2013, 34(7), 1731—1738)
[5]	Peterson T. R., Laplante M., Thoreen C. C., Sancak Y., Kang S. A., Kuehl W. M., Gray N. S., Sabatini D. M., Cell, 2009, 137(5), 873—886
[6]	Fingar D. C., Salama S., Tsou C., Harlow E., Belnis J., GenesDev., 2002, 16(12), 1472—1487
[7]	Gera J., Lichtenstein A., LeukLymphoma, 2011, 52(10), 1857—1866
[8]	Lu M., Wang J., Elves H., Pearce D., J. Biol.Chem., 2011, 286(35), 30647—30654
[9]	Chen J., Zheng X. F., Brown E. J., Scheriber S. L., Proc. Natl. Acad.Sci., 1995, 92(11), 4947—4951
[10]	Yang H., Rudge D. G., Koos J. D., Vaidialingam B., Yang H. J., Pavletich N. P., Nature,2013, 497(7448), 217—223
[11]	Dames S. A., J. Biol.Chem., 2010, 285(10), 7766—7775
[12]	Hardt M., Chantaravisoot N., Tamanoi F., Genes toCells, 2011, 16(2), 141—151
[13]	Sturgill T. W., Hall M. N., ACS Chem.Biol., 2009, 4(12), 999—1015
[14]	Bakkenist C. J., Kastan M. B., Nature,2003, 421(6922), 499—506
[15]	Mehtab P., Mohammed M. A., Akhtar A., Mahboob A., Chem. Res. ChineseUniversities, 2014, 30(1), 55—62
[16]	Imamura S., Kishi S., Int. J. Biochem. Cell. Biol., 2005, 37(5), 1105—1116
[17]	Tanaka A., Weinel S., Nagy N., O’Driscoll M., Lai-Cheong J. E., Kulp-Shorten C. L., Knable A., Carpenter G., Fisher S. A., Hiragun M., Yanase Y., Hide M., Callen J., McGrath J. A., Am. J. Hum.Genet., 2012, 90(3), 511—517
[18]	Jiang X., Sun Y., Chen S., Roy K., Price B. D., J. Biol.Chem., 2006, 281(23), 15741—15746
[19]	Rivera-Calzada A., Maman J. D., Spagnolo L., Pearl L. H., Llorca O., Structure, 2005, 13(2), 243—255
[20]	de la Rubia J., Such E., LeukLymphoma, 2010, 51(11), 1960—1961
[21]	Tsai W. B., Chung Y. M., Takahashi Y., Xu Z., Hu M. C., Nat. Cell.Biol., 2008, 10(4), 460—467
[22]	Kazi A. A., Hong-Brown L., Lang S. M., Lang C. H., Mol.Med., 2011, 17(9), 925—936
[23]	Zhang X. Y., Deng D. J., Tan J. J., He Y., Li C. H., Wang C. X., Chem. Res. ChineseUniversities, 2014, 30(2), 297—305