Department of Chemical Engineering, University of Washington, Seattle, Washington.
Biotechnol Bioeng. 2019 Apr;116(4):912-918. doi: 10.1002/bit.26878. Epub 2018 Dec 31.
Blue-absorbing proteorhodopsin (BPR) from marine bacteria is a retinal-bound, light-activated, outwards proton transporter containing seven α-helical transmembrane segments (TMS). It is synthesized as a precursor species (pre-BPR) with a predicted N-terminal signal sequence that is cleaved to yield the mature protein. While optimizing the production of BPR in Escherichia coli to facilitate the construction of bioprotonic devices, we observed significant pre-BPR accumulation in the inner membrane and explored signal sequence requirements and export pathway. We report here that BPR does not rely on the Sec pathway for inner membrane integration, and that although it greatly enhances yields, its signal sequence is not necessary to obtain a functional product. We further show that an unprocessable version of pre-BPR obtained by mutagenesis of the signal peptidase I site exhibits all functional attributes of the wild-type protein and has the advantage of being produced at higher levels. Our results are consistent with the BPR signal sequence being recognized by the signal recognition particle (SRP; a protein that orchestrates the cotranslational biogenesis of inner membrane proteins) and serving as a beneficial "pro" domain rather than a traditional secretory peptide.
海洋细菌中的蓝吸收蛋白视紫红质（BPR）是一种结合视黄醛的、光激活的外向质子转运体，含有七个α-螺旋跨膜片段（TMS）。它作为一种前体物质（前 BPR）合成，预测有一个 N 端信号序列，该序列被切割后产生成熟蛋白。在优化大肠杆菌中 BPR 的生产以促进生物质子器件的构建过程中，我们观察到前 BPR 在内膜中大量积累，并探索了信号序列要求和输出途径。我们在这里报告，BPR 不依赖于 Sec 途径进行内膜整合，虽然它极大地提高了产量，但它的信号序列对于获得功能性产物并不是必需的。我们进一步表明，通过突变信号肽酶 I 位点获得的不可加工的前 BPR 版本表现出与野生型蛋白相同的所有功能属性，并且具有在更高水平上生产的优势。我们的结果与 BPR 信号序列被信号识别颗粒（SRP；一种协调内膜蛋白共翻译生物发生的蛋白质）识别并作为有益的“前导”结构域而不是传统分泌肽的观点一致。
