BACKGROUND Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure. RESULTS Transfection conditions were first optimized to achieve expression levels between 10 and 18 mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4-9 mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure ( ≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (Kd) of 5 nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured kcat (7.2 ± 0.5 min- 1) and KM (1.2 ± 0.3 μM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption. CONCLUSIONS The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher kcat and KM values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway.

中文翻译：

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通过转染悬浮液中培养的人HEK293-EBNA1细胞，可高产生产人Dicer。

背景技术Dicer是一种219 kDa蛋白，在基因调控中起着关键作用，特别是作为负责裂解前体miRNA底物的核糖核酸酶III酶。它的酶促活性受到蛋白质因子的高度调节，这种调节可影响miRNA的水平并调节细胞的行为。为了更好地理解调节的基本机制，需要对Dicer进行详细的酶和结构表征。但是，这些类型的研究通常需要几毫克的重组蛋白，而有效制备如此数量的纯人Dicer仍然是一个挑战。为了制备大量的人Dicer，我们优化了悬浮培养的HEK293-6E细胞的转染，并简化了纯化程序。结果首先优化转染条件，使每升培养物的表达量达到10至18 mg重组Dicer。然后开发了三步纯化方案，可在一天之内每升培养物产生4-9 mg纯化的Dicer。通过SEC-MALS / RI分析和TEM阴性染色，我们证实了纯化的蛋白质是单体纯净的（≥98％），并具有特征性的L形折叠。与先前的报道一致，使用电泳迁移率变动测定法，测得Dicer与pre-let-7a-1的结合的解离常数（Kd）为5 nM。但是，在探索Dicer对pre-let-7a-1的切割活性时，我们测量了kcat（7.2±0.5 min-1）和KM（1.2±0）。3μM）的值比以前报道的要高得多，原因是实验条件更好地符合了稳态假设。结论本文所述的表达和纯化方案可提供高产率的单体纯净且有活性的人Dicer。用该纯化的切丁酶对pre-let-7底物进行的切割研究显示，其kcat和KM值比以前报道的要高，并且支持当前的观点，即构象变化与底物结合有关。大量的高纯度Dicer将对miRNA途径这一关键蛋白质的未来生化，生物物理和结构研究具有价值。用该纯化的切丁酶对pre-let-7底物进行的切割研究显示，其kcat和KM值比以前报道的要高，并且支持当前的观点，即构象变化与底物结合有关。大量的高纯度Dicer将对miRNA途径这一关键蛋白质的未来生化，生物物理和结构研究具有价值。用该纯化的切丁酶对pre-let-7底物进行的切割研究显示，其kcat和KM值比以前报道的要高，并且支持当前的观点，即构象变化与底物结合有关。大量的高纯度Dicer将对miRNA途径这一关键蛋白质的未来生化，生物物理和结构研究具有价值。