Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, USA.
Biochemistry. 2012 Jun 19;51(24):4822-34. doi: 10.1021/bi300412y. Epub 2012 Jun 8.
The periplasmic seventeen kilodalton protein (Skp) chaperone has been characterized primarily for its role in outer membrane protein (OMP) biogenesis, during which the jellyfish-like trimeric protein encapsulates partially folded OMPs, protecting them from the aqueous environment until delivery to the BAM outer membrane protein insertion complex. However, Skp is increasingly recognized as a chaperone that also assists in folding soluble proteins in the bacterial periplasm. In this capacity, Skp coexpression increases the active yields of many recombinant proteins and bacterial virulence factors. Using a panel of single-chain antibodies and a single-chain T-cell receptor (collectively termed scFvs) possessing varying stabilities and biophysical characteristics, we performed in vivo expression and in vitro folding and aggregation assays in the presence or absence of Skp. For Skp-sensitive scFvs, the presence of Skp during in vitro refolding assays reduced aggregation but did not alter the observed folding rates, resulting in a higher overall yield of active protein. Of the proteins analyzed, Skp sensitivity in all assays correlated with the presence of folding intermediates, as observed with urea denaturation studies. These results are consistent with Skp acting as a holdase, sequestering partially folded intermediates and thereby preventing aggregation. Because not all soluble proteins are sensitive to Skp coexpression, we hypothesize that the presence of a long-lived protein folding intermediate renders a protein sensitive to Skp. Improved understanding of the bacterial periplasmic protein folding machinery may assist in high-level recombinant protein expression and may help identify novel approaches to block bacterial virulence.
周质十七 kDa 蛋白 (Skp) 伴侣主要因其在外膜蛋白 (OMP) 生物发生过程中的作用而被描述，在此过程中，水母状三聚体蛋白将部分折叠的 OMP 包裹起来，使其免受水相环境的影响，直到递送至 BAM 外膜蛋白插入复合物。然而，Skp 越来越被认为是一种伴侣，它还协助折叠细菌周质中的可溶性蛋白。在这种情况下，Skp 的共表达增加了许多重组蛋白和细菌毒力因子的活性产量。使用一组单链抗体和单个单链 T 细胞受体（统称为 scFvs），它们具有不同的稳定性和生物物理特性，我们在存在或不存在 Skp 的情况下进行了体内表达和体外折叠和聚集测定。对于对 Skp 敏感的 scFvs，在体外重折叠测定中存在 Skp 会减少聚集，但不会改变观察到的折叠速率，从而导致更高的活性蛋白总产量。在所分析的蛋白质中，所有测定中 Skp 敏感性都与折叠中间体的存在相关，如尿素变性研究中观察到的那样。这些结果与 Skp 作为持留蛋白的作用一致，它会隔离部分折叠的中间体，从而防止聚集。由于并非所有可溶性蛋白都对 Skp 共表达敏感，我们假设存在长寿的蛋白质折叠中间体使蛋白质对 Skp 敏感。对细菌周质蛋白折叠机制的更好理解可能有助于高水平的重组蛋白表达，并有助于识别阻止细菌毒力的新方法。
