Kolb V A, Makeyev E V, Spirin A S
Institute of Protein Research, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia.
J Biol Chem. 2000 Jun 2;275(22):16597-601. doi: 10.1074/jbc.M002030200.
Continuous monitoring of the enzymatic activity of newly synthesized firefly luciferase in Escherichia coli cell-free translation system was performed to record folding kinetics of this multidomain eukaryotic protein in the prokaryotic cytosol. Whereas in vitro refolding of denatured luciferase in prokaryotic cytosol occurred with a low yield of active enzyme and took about an hour, the enzyme acquired its native structure immediately upon release from the ribosome, as seen from the immediate halt of active luciferase accumulation upon blocking of translation with inhibitors. The nascent luciferase was also capable of acquiring the active conformation prior to release from the ribosome, when its C terminus was extended with a polypeptide segment. Specific enzymatic activity of the firefly luciferase was found to be equally high irrespective of whether this protein was synthesized in eukaryotic or prokaryotic translation systems. The data presented demonstrate the fundamental ability of prokaryotic cytosol to support effective co-translational protein folding in general and co-translational folding of multidomain proteins in particular.
在大肠杆菌无细胞翻译系统中对新合成的萤火虫荧光素酶的酶活性进行连续监测，以记录这种多结构域真核蛋白在原核细胞质中的折叠动力学。虽然变性荧光素酶在原核细胞质中的体外重折叠产生的活性酶产量较低且耗时约一小时，但从用抑制剂阻断翻译后活性荧光素酶积累立即停止可以看出，该酶从核糖体释放后立即获得其天然结构。当新生荧光素酶的C末端用一个多肽片段延伸时，它在从核糖体释放之前也能够获得活性构象。无论该蛋白是在真核还是原核翻译系统中合成，萤火虫荧光素酶的比酶活性都同样高。所呈现的数据表明，原核细胞质一般具有支持有效的共翻译蛋白质折叠的基本能力，特别是多结构域蛋白质的共翻译折叠能力。
