Braud Sandrine, Moutiez Mireille, Belin Pascal, Abello Nicolas, Drevet Pascal, Zinn-Justin Sophie, Courçon Marie, Masson Cédric, Dassa Janie, Charbonnier Jean-Baptiste, Boulain Jean-Claude, Ménez André, Genet Roger, Gondry Muriel
CEA/Saclay, Département d'Ingénierie et d'Etudes des Protéines, F-91191 Gif-sur-Yvette Cedex, France.
J Proteome Res. 2005 Nov-Dec;4(6):2137-47. doi: 10.1021/pr050230i.
Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 microL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S.tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.
许多旨在从结构或功能上表征蛋白质组的研究需要以高通量形式生产纯蛋白质。我们开发了一种快速且灵活的集成系统，用于克隆、在大肠杆菌中表达蛋白质、进行溶解度筛选和纯化，该系统可以在96孔微孔板中完全自动化操作。我们在定制设计的载体中使用重组克隆，这些载体包括：（i）一个编码（His）6标签的序列；（ii）一个可变的增溶伴侣基因；（iii）对应于TEV蛋白酶切割位点的DNA序列；（iv）感兴趣的基因（或DNA片段）；（v）一个可抑制的琥珀色终止密码子；（vi）一个编码S.tag肽的序列。首先，优化了微孔板（250 μL）中细菌培养的条件，以获得与1-L烧瓶（100-mL培养物）中相同的表达和溶解度模式。这些条件除了能筛选融合伴侣外，还能筛选各种参数（大肠杆菌菌株、温度、诱导剂等）。其次，在琥珀色抑制菌株中表达融合蛋白，通过检测S.tag，利用荧光对可溶性和不溶性蛋白进行定量。该技术比其他常用方法（斑点印迹、蛋白质免疫印迹、SDS-PAGE）更快、更灵敏。结果表明，琥珀色抑制tRNA的存在既不影响靶蛋白的表达模式，也不影响其溶解度。第三，通过我们的筛选方法检测到的最有趣的可溶性融合蛋白，可以在非抑制菌株中生产。用TEV蛋白酶切割后，可获得具有独特额外N端甘氨酸的天然形式的靶蛋白。
