Löw Christian, Moberg Per, Quistgaard Esben M, Hedrén Marie, Guettou Fatma, Frauenfeld Jens, Haneskog Lars, Nordlund Pär
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Biochim Biophys Acta. 2013 Jun;1830(6):3497-508. doi: 10.1016/j.bbagen.2013.02.001. Epub 2013 Feb 9.
Structural studies of integral membrane proteins (IMPs) are often hampered by difficulties in producing stable homogenous samples for crystallization. To overcome this hurdle it has become common practice to screen large numbers of target proteins to find suitable candidates for crystallization. For such an approach to be effective, an efficient screening strategy is imperative. To this end, strategies have been developed that involve the use of green fluorescent protein (GFP) fusion constructs. However, these approaches suffer from two drawbacks; proteins with a translocated C-terminus cannot be tested and scale-up from analytical to preparative purification is often non-trivial and may require re-cloning.
Here we present a screening approach that prioritizes IMP targets based on three criteria: expression level, detergent solubilization yield and homogeneity as determined by high-throughput small-scale immobilized metal affinity chromatography (IMAC) and automated size-exclusion chromatography (SEC).
To validate the strategy, we screened 48 prokaryotic IMPs in two different vectors and two Escherichia coli strains. A set of 11 proteins passed all preset quality control checkpoints and was subjected to crystallization trials. Four of these crystallized directly in initial sparse matrix screens, highlighting the robustness of the strategy.
We have developed a rapid and cost efficient screening strategy that can be used for all IMPs regardless of topology. The analytical steps have been designed to be a good mimic of preparative purification, which greatly facilitates scale-up.
The screening approach presented here is intended and expected to help drive forward structural biology of membrane proteins.
整合膜蛋白（IMP）的结构研究常常因难以制备用于结晶的稳定均一样品而受阻。为克服这一障碍，大量筛选目标蛋白以寻找适合结晶的候选蛋白已成为常见做法。要使这种方法有效，高效的筛选策略至关重要。为此，已开发出涉及使用绿色荧光蛋白（GFP）融合构建体的策略。然而，这些方法存在两个缺点：无法测试C端易位的蛋白质，并且从分析纯化扩大到制备纯化通常并非易事，可能需要重新克隆。
在此，我们提出一种筛选方法，该方法基于三个标准对IMP目标进行优先级排序：表达水平、去污剂增溶产量以及通过高通量小规模固定金属亲和色谱（IMAC）和自动尺寸排阻色谱（SEC）测定的均一性。
为验证该策略，我们在两种不同载体和两种大肠杆菌菌株中筛选了48种原核IMP。一组11种蛋白质通过了所有预设的质量控制检查点，并进行了结晶试验。其中四种在初始稀疏矩阵筛选中直接结晶，突出了该策略的稳健性。
我们开发了一种快速且经济高效的筛选策略，可用于所有拓扑结构的IMP。分析步骤设计为很好地模拟制备纯化，这极大地促进了放大过程。
本文提出的筛选方法旨在并有望推动膜蛋白的结构生物学研究。
