Cloning, expression and purification of the low-complexity region of RanBP9 protein
Cloning, expression and purification of the low-complexity region of RanBP9 protein
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DOI：
10.1016/j.pep.2020.105630
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发表时间：
2020-08-01
影响因子：
1.6
通讯作者：
Rangachari, Vijayaraghavan
中科院分区：
文献类型：
--
作者：
Dhakal, Shailendra;Sapkota, Krishna;Rangachari, Vijayaraghavan
蛋白质的重组表达和纯化是生物化学和生物物理研究的关键。虽然这已经成为许多蛋白质的常规和标准程序，但本质上无序的蛋白质和具有低复杂性序列的蛋白质造成了困难。含有低复杂性区域（LCR）的蛋白质在正常和病理过程中的作用越来越重要。本文报道了RanBP 9蛋白N端LCR的克隆、表达和纯化。RanBP 9是存在于细胞质和细胞核中的支架蛋白，其涉及许多细胞过程。Nt-RanBP 9是该蛋白质的一个知之甚少的区域，这可能是由于LCR带来的困难。实际上，由于Nt-RanBP 9的高GC含量导致不显著的蛋白质表达，常规方法在Nt-RanBP 9克隆中存在困难。这些导致我们使用不同的克隆方法，通过使用体内DNA重组方法将蛋白质表达为含有mCherry或mEGFP的融合构建体。我们的结果表明，表达mEGFP标记的Nt-RanBP 9，然后凝血酶切割标签是获得纯度> 90%且产率良好的蛋白质的最有效方法。我们报告并讨论了获得RanBP 9的N-末端区域的挑战，RanBP 9是一种在多种生物过程和神经退行性疾病中具有功能意义的蛋白质。
Recombinant expression and purification of proteins is key for biochemical and biophysical investigations. Although this has become a routine and standard procedure for many proteins, intrinsically disordered ones and those with low complexity sequences pose difficulties. Proteins containing low complexity regions (LCRs) are increasingly becoming significant for their roles in both normal and pathological processes. Here, we report cloning, expression and purification of N-terminal LCR of RanBP9 protein (Nt-RanBP9). RanBP9 is a scaffolding protein present in both cytoplasm and nucleus that is implicated in many cellular processes. Nt-RanBP9 is a poorly understood region of the protein perhaps due to difficulties posed by the LCR. Indeed, conventional methods presented difficulties in Nt-RanBP9 cloning due to its high GC content resulting in insignificant protein expression. These led us to use a different approach of cloning by expressing the protein as a fusion construct containing mCherry or mEGFP using in vivo DNA recombination methods. Our results indicate that expression of mEGFP-tagged Nt-RanBP9 followed by thrombin cleavage of the tag was the most effective method to obtain the protein with > 90% purity and good yields. We report and discuss the challenges in obtaining the N-terminal region of RanBP9, a protein with functional implications in multiple biological processes and neurodegenerative diseases.