Hillebrecht Jason R, Chong Shaorong
New England Biolabs, 240 County Road, Ipswich, MA 01938, USA.
BMC Biotechnol. 2008 Jul 29;8:58. doi: 10.1186/1472-6750-8-58.
Cell-free protein synthesis is not only a rapid and high throughput technology to obtain proteins from their genes, but also provides an in vitro platform to study protein translation and folding. A detailed comparison of in vitro protein synthesis in different cell-free systems may provide insights to their biological differences and guidelines for their applications.
Protein synthesis was investigated in vitro in a reconstituted prokaryotic system, a S30 extract-based system and a eukaryotic system. Compared to the S30 system, protein synthesis in the reconstituted system resulted in a reduced yield, and was more cold-sensitive. Supplementing the reconstituted system with fractions from a size-exclusion separation of the S30 extract significantly increased the yield and activity, to a level close to that of the S30 system. Though protein synthesis in both prokaryotic and eukaryotic systems showed no significant differences for eukaryotic reporter proteins, drastic differences were observed when an artificial fusion protein was synthesized in vitro. The prokaryotic systems failed to synthesize and correctly fold a significant amount of the full-length fusion protein, even when supplemented with the eukaryotic lysate. The active full-length fusion protein was synthesized only in the eukaryotic system.
The reconstituted bacterial system is sufficient but not efficient in protein synthesis. The S30 system by comparison contains additional cellular factors capable of enhancing protein translation and folding. The eukaryotic translation machinery may have evolved from its prokaryotic counterpart in order to translate more complex (difficult-to-translate) templates into active proteins.
无细胞蛋白质合成不仅是一种从基因中获取蛋白质的快速且高通量的技术，还提供了一个研究蛋白质翻译和折叠的体外平台。对不同无细胞系统中的体外蛋白质合成进行详细比较，可能有助于深入了解它们的生物学差异，并为其应用提供指导。
在体外对重组原核系统、基于S30提取物的系统和真核系统中的蛋白质合成进行了研究。与S30系统相比，重组系统中的蛋白质合成产量降低，且对低温更敏感。用S30提取物经尺寸排阻分离得到的组分补充重组系统，可显著提高产量和活性，使其接近S30系统的水平。尽管原核和真核系统中对真核报告蛋白的蛋白质合成没有显著差异，但在体外合成人工融合蛋白时观察到了显著差异。即使补充了真核裂解物，原核系统也无法合成并正确折叠大量全长融合蛋白。只有在真核系统中才能合成有活性的全长融合蛋白。
重组细菌系统在蛋白质合成方面是足够的，但效率不高。相比之下，S30系统含有能够增强蛋白质翻译和折叠的额外细胞因子。真核翻译机制可能是从原核对应物进化而来的，以便将更复杂（难以翻译）的模板翻译成活性蛋白。
