<br>通过体内 BirA 介导的生物素化从果蝇卵巢和胚胎中分离分泌蛋白。,PLOS ONE

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通过体内 BirA 介导的生物素化从果蝇卵巢和胚胎中分离分泌蛋白。
PLOS ONE ( IF 2.9 ) Pub Date : 2019-10-28 , DOI: 10.1371/journal.pone.0219878
Leslie M Stevens _{1,

2} , Yuan Zhang ₂ , Yuri Volnov ₁ , Geng Chen _{1,

2} , David S Stein _{1,

2}

Affiliation

生物素和亲和素之间异常强烈的非共价相互作用 (kD = 10-14-10-16) 使得这种相互作用能够在各种实验环境中使用。生物素受体肽 (BAP) 是一个 15 个氨基酸基序，可被大肠杆菌 BirA 蛋白生物素化，已与目标蛋白融合，使其成为体内生物素化的底物。在这里，我们报告了修饰的 BirA 的构建和表征，该 BirA 带有用于分泌和内质网 (ER) 保留的信号，用于需要分泌蛋白生物素化的实验环境。当在 Gal4 介导的转录控制下在果蝇雌性种系或卵泡细胞中表达时，可以检测到修饰的 BirA 蛋白，并显示其在卵巢和子代胚胎中具有酶活性。然而，令人惊讶的是，它并没有有效地保留在内质网中，而是似乎被分泌出来。为了确定这种分泌蛋白（现在称为 secBirA）是否可以对分泌蛋白进行生物素化，我们生成了两种果蝇分泌蛋白的 BAP 标记版本：躯干样蛋白 (Tsl) 和原肠胚形成缺陷蛋白 (GD)，这两种蛋白通常在母体表达并参与胚胎模式形成。 Tsl-BAP 和 GD-BAP 均显示出正常的图案形成活性。 Tsl-BAP 与 secBirA 在卵巢卵泡细胞中的共表达导致其生物素化，这使得可以使用亲和素偶联的亲和基质将其从卵巢和子代胚胎中分离出来。相反，在雌性种系中与 secBirA 共表达不会导致可检测到的 GD-BAP 生物素化，可能是因为 BAP 标签的 C 端位置使其无法在体内与 BirA 接触。我们的结果表明，secBirA 指导体内分泌结合蛋白的生物素化，为感兴趣的分泌蛋白提供了强大的实验方法。然而，目标蛋白的有效生物素化可能会因 BAP 标签的位置或蛋白的其他结构特征而异。

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Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation.

The extraordinarily strong non-covalent interaction between biotin and avidin (kD = 10-14-10-16) has permitted this interaction to be used in a wide variety of experimental contexts. The Biotin Acceptor Peptide (BAP), a 15 amino acid motif that can be biotinylated by the E. coli BirA protein, has been fused to proteins-of-interest, making them substrates for in vivo biotinylation. Here we report on the construction and characterization of a modified BirA bearing signals for secretion and endoplasmic reticulum (ER) retention, for use in experimental contexts requiring biotinylation of secreted proteins. When expressed in the Drosophila female germline or ovarian follicle cells under Gal4-mediated transcriptional control, the modified BirA protein could be detected and shown to be enzymatically active in ovaries and progeny embryos. Surprisingly, however, it was not efficiently retained in the ER, and instead appeared to be secreted. To determine whether this secreted protein, now designated secBirA, could biotinylate secreted proteins, we generated BAP-tagged versions of two secreted Drosophila proteins, Torsolike (Tsl) and Gastrulation Defective (GD), which are normally expressed maternally and participate in embryonic pattern formation. Both Tsl-BAP and GD-BAP were shown to exhibit normal patterning activity. Co-expression of Tsl-BAP together with secBirA in ovarian follicle cells resulted in its biotinylation, which permitted its isolation from both ovaries and progeny embryos using Avidin-coupled affinity matrix. In contrast, co-expression with secBirA in the female germline did not result in detectable biotinylation of GD-BAP, possibly because the C-terminal location of the BAP tag made it inaccessible to BirA in vivo. Our results indicate that secBirA directs biotinylation of proteins bound for secretion in vivo, providing access to powerful experimental approaches for secreted proteins-of-interest. However, efficient biotinylation of target proteins may vary depending upon the location of the BAP tag or other structural features of the protein.

更新日期：2019-10-28

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