SUMO作为一种溶解性标签以及SUMO融合蛋白与Ulp1的体内切割。
SUMO as a solubility tag and in vivo cleavage of SUMO fusion proteins with Ulp1.
作者信息
Kuo Dennis, Nie Minghua, Courey Albert J
机构信息
Department of Chemistry & Biochemistry, Molecular Biology Institute, University of California, 607 Charles E. Young Drive, East, Los Angeles, CA, 90095-1569, USA.
出版信息
Methods Mol Biol. 2014;1177:71-80. doi: 10.1007/978-1-4939-1034-2_6.
Expression of proteins in E. coli is often plagued by insolubility of the protein of interest. A solution to this problem is the expression of proteins as fusions to solubility tags such as the SUMO protein. SUMO fusion proteins can be cleaved to remove the SUMO moiety using SUMO-specific proteases such as Ulp1. Here, we describe the use of vectors for the expression of recombinant proteins in E. coli as fusions to the Drosophila SUMO protein. This includes a vector that encodes not only the SUMO tagged protein of interest but also SUMO-tagged Ulp1. Coexpression of these two proteins results in the in vivo cleavage of the protein of interest from the SUMO tag, while still leaving the protein of interest in a form that can be purified from a soluble cell lysate by nickel affinity chromatography.
在大肠杆菌中表达蛋白质时，目标蛋白的不溶性常常困扰着实验。解决这个问题的一个办法是将蛋白质与溶解性标签（如SUMO蛋白）融合表达。SUMO融合蛋白可以使用SUMO特异性蛋白酶（如Ulp1）进行切割以去除SUMO部分。在此，我们描述了用于在大肠杆菌中表达重组蛋白并使其与果蝇SUMO蛋白融合的载体的使用。这包括一种不仅编码感兴趣的SUMO标签蛋白，还编码SUMO标签的Ulp1的载体。这两种蛋白的共表达导致目标蛋白在体内从SUMO标签上切割下来，同时仍使目标蛋白保持一种可以通过镍亲和层析从可溶性细胞裂解物中纯化的形式。
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