Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stanford, California 94305, USA.
Biotechnol Bioeng. 2011 Aug;108(8):1739-48. doi: 10.1002/bit.23111. Epub 2011 Mar 21.
The endoplasmic reticulum (ER) resident Hsp70 chaperone, BiP, docks to the Sec translocon and interacts co-translationally with polypeptides entering the ER to encourage proper folding. In order to recreate this interaction in Escherichia coli cell-free protein synthesis (CFPS) reactions, a fusion protein was formed between the ribosome-binding portion of the E. coli protein trigger factor (TF) and BiP. The biophysical affinity to ribosomes as well as the characteristic Hsp70 ATPase activity were both verified for the fusion protein. When added to E. coli-based CFPS reactions, the TF-BiP fusion chaperone increased soluble yields of several protein fragments that are normally secreted through the ER and have poor solubility in typical CFPS reactions. For comparison, a fusion between TF and the native E. coli Hsp70, DnaK, was also constructed. This fusion was also biologically active and increased soluble yields of certain protein targets in CFPS. The TF-BiP fusion described in this study can be seen as a first step in reconstituting and better understanding ER folding pathways in the prokaryotic environment of E. coli CFPS.
内质网（ER）驻留的 Hsp70 伴侣蛋白 BiP 与 Sec 易位子结合，并与进入 ER 的多肽进行共翻译相互作用，以促进正确折叠。为了在大肠杆菌无细胞蛋白合成（CFPS）反应中重现这种相互作用，在大肠杆菌蛋白触发因子（TF）的核糖体结合部分和 BiP 之间形成融合蛋白。融合蛋白的生物物理亲和力以及特征性的 Hsp70 ATP 酶活性都得到了验证。当添加到基于大肠杆菌的 CFPS 反应中时，TF-BiP 融合伴侣蛋白增加了几种通常通过 ER 分泌且在典型 CFPS 反应中溶解度较差的蛋白片段的可溶性产量。相比之下，还构建了 TF 与天然大肠杆菌 Hsp70、DnaK 之间的融合。该融合也具有生物活性，并增加了 CFPS 中某些蛋白靶标的可溶性产量。本研究中描述的 TF-BiP 融合蛋白可以被视为在大肠杆菌 CFPS 的原核环境中重新构建和更好地理解 ER 折叠途径的第一步。
