Sonar S, Patel N, Fischer W, Rothschild K J
Physics Department, Boston University, Massachusetts 02215.
Biochemistry. 1993 Dec 21;32(50):13777-81. doi: 10.1021/bi00213a004.
Bacteriorhodopsin (bR) is an integral membrane protein which functions as a light-driven proton pump in Halobacterium halobium (also known as Halobacterium salinarium). The cell-free synthesis of bR in quantities sufficient for FTIR and NMR spectroscopy and the ability to selectively isotope label bR using aminoacylated suppressor tRNAs would provide a powerful approach for studying the role of specific amino acid residues. However, no integral membrane protein has yet been expressed in a cell-free system in quantities sufficient for such biophysical studies. We report the cell-free synthesis of bacterioopsin, its purification, its refolding in polar lipids from H. halobium, and its regeneration with all-trans-retinal to yield bacteriorhodopsin in a form functionally similar to bR in purple membrane. Importantly, the yields obtained from in vitro and in vivo expression are comparable. Functionality of the cell-free expressed bR is established using static and time-resolved absorption spectroscopy and FTIR difference spectroscopy.
细菌视紫红质（bR）是一种整合膜蛋白，在嗜盐菌（也称为盐生盐杆菌）中作为光驱动质子泵发挥作用。以足以用于傅里叶变换红外光谱（FTIR）和核磁共振光谱（NMR）的量在无细胞体系中合成bR，以及使用氨酰化抑制tRNA对bR进行选择性同位素标记的能力，将为研究特定氨基酸残基的作用提供一种强大的方法。然而，尚未有整合膜蛋白在无细胞体系中以足以进行此类生物物理研究的量表达。我们报道了细菌视蛋白在无细胞体系中的合成、纯化、在来自嗜盐菌的极性脂质中重折叠，以及用全反式视黄醛再生以产生在功能上类似于紫色膜中bR的细菌视紫红质。重要的是，体外和体内表达获得的产量相当。使用静态和时间分辨吸收光谱以及FTIR差分光谱确定了无细胞表达的bR的功能。
