Expression and purification of full-length mouse CARM1 from transiently transfected HEK293T cells using HaloTag technology.
Expression and purification of full-length mouse CARM1 from transiently transfected HEK293T cells using HaloTag technology.
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DOI：
10.1016/j.pep.2010.11.010
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发表时间：
2011-04
影响因子：
1.6
通讯作者：
Burgess, Richard R.
中科院分区：
文献类型：
--
作者：
Chumanov, Robert S.;Kuhn, Peter A.;Xu, Wei;Burgess, Richard R.
辅激活因子相关精氨酸甲基转移酶1（CARM 1）是蛋白质精氨酸甲基转移酶（PRMT）家族成员，在雄激素和雌激素信号通路中起辅激活剂的作用，并在前列腺癌和乳腺癌的进展中发挥作用。CARM 1催化多种蛋白质底物的甲基化。先前纯化全长小鼠CARM 1蛋白的尝试已被证明是不令人满意的。在E.大肠杆菌形成难以变性和再折叠的不溶性包涵体。所呈现的结果证明了使用新的HaloTag™技术从大肠杆菌和大肠杆菌纯化全长CARM 1。coli和哺乳动物HEK 293 T细胞。从E.然而，该蛋白质在N-末端被截短了10-50个氨基酸，最可能是由于内源性蛋白水解活性。相反，从瞬时转染的HEK 293 T细胞中纯化了大量的可溶性全长CARM 1。来自HEK 293 T细胞的CARM 1与许多共纯化的相互作用蛋白一起分离。HaloTag和HaloLink树脂之间形成的共价键允许使用严格的洗涤条件，而没有洗脱CARM 1蛋白的风险。结果还说明了一种用于纯化和富集CARM 1相关蛋白以及CARM 1甲基转移酶活性底物的高效方法。
Coactivator-associated arginine methyl transferase 1 (CARM1) is a protein arginine methyltransferase (PRMT) family member that functions as a coactivator in androgen and estrogen signaling pathways and plays a role in the progression of prostate and breast cancer. CARM1 catalyzes methylation of diverse protein substrates. Prior attempts to purify the full-length mouse CARM1 protein have proven unsatisfactory. The full-length protein expressed in E. coli forms insoluble inclusion bodies that are difficult to denature and to refold. The presented results demonstrate the use of a novel HaloTag™ technology to purify full-length CARM1 from both E. coli and mammalian HEK293T cells. A small amount of CARM1 was purified from E. coli; however, the protein was truncated on the N-terminus by 10–50 amino acids, most likely due to endogenous proteolytic activity. In contrast, substantial quantities of soluble full-length CARM1 were purified from transiently transfected HEK293T cells. The CARM1 from HEK293T cells was isolated alongside a number of co-purifying interacting proteins. The covalent bond formed between the HaloTag and the HaloLink resin allowed the use of stringent wash conditions without risk of eluting the CARM1 protein. The results also illustrate a highly effective approach for purifying and enriching both CARM1-associated proteins as well as substrates for CARM1’s methyltransferase activity.
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DOI：
10.1073/pnas.0610792104
发表时间：
2007-07-24
影响因子：
11.1
作者：
Higashimoto, Ken;Kuhn, Peter;Xu, Wei
通讯作者：
Xu, Wei
影响因子：
4
作者：
Bedford, Mark T.
通讯作者：
Bedford, Mark T.
影响因子：
56.9
作者：
Xu, W;Chen, HW;Evans, RM
通讯作者：
Evans, RM
影响因子：
1.6
作者：
Chumanov, Robert S.;Burgess, Richard R.
通讯作者：
Burgess, Richard R.
影响因子：
--
作者：
Teyssier, Catherine;Ou, Chen-Yin;Stallcup, Michael R.
通讯作者：
Stallcup, Michael R.