Ideno A, Furutani M, Iwabuchi T, Iida T, Iba Y, Kurosawa Y, Sakuraba H, Ohshima T, Kawarabayashi Y, Maruyama T
Marine Biotechnology Institute, Kamaishi, 026-0001 Iwate, Japan.
Appl Microbiol Biotechnol. 2004 Mar;64(1):99-105. doi: 10.1007/s00253-003-1459-4. Epub 2003 Oct 15.
Improper protein-folding often results in inclusion-body formation in a protein expression system using Escherichia coli. To express such proteins in the soluble fraction of E. coli cytoplasm, we developed an expression system by fusing the target protein with an archaeal FK506 binding protein (FKBP). It has been reported that an archaeal FKBP from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TcFKBP18), possesses not only peptidyl-prolyl cis-trans isomerase activity, but also chaperone-like activity to enhance the refolding yield of an unfolded protein by suppressing irreversible protein aggregation. To study the effect of this fusion strategy with FKBP on the expression of foreign protein in E. coli, a putative rhodanese (thiosulfate sulfurtransferase) from a hyperthermophilic archaeon and two mouse antibody fragments were used as model target proteins. When they were expressed alone in E. coli, they formed insoluble aggregates. Their genes were designed to be expressed as a fusion protein by connecting them to the C-terminal end of TcFKBP18 with an oligopeptide containing a thrombin cleavage site. By fusing TcFKBP18, the expression of the target protein in the soluble fraction was significantly increased. The percentage of the soluble form in the expressed protein reached 10-28% of the host soluble proteins. After purification and protease digestion of the expressed antibody fragment-TcFKBP18 fusion protein, the cleaved antibody fragment (single-chain Fv) showed specific binding to the antigen in ELISA. This indicated that the expressed antibody fragment properly folded to the active form.
在使用大肠杆菌的蛋白质表达系统中，蛋白质折叠不当常常导致包涵体形成。为了在大肠杆菌细胞质的可溶部分表达此类蛋白质，我们通过将目标蛋白与古菌FK506结合蛋白（FKBP）融合，开发了一种表达系统。据报道，来自嗜热古菌嗜热栖热菌KS-1（TcFKBP18）的古菌FKBP不仅具有肽基脯氨酰顺反异构酶活性，还具有伴侣样活性，可通过抑制不可逆的蛋白质聚集来提高未折叠蛋白的重折叠产率。为了研究这种与FKBP的融合策略对大肠杆菌中外源蛋白表达的影响，我们使用了来自嗜热古菌的一种假定硫氰酸酶（硫代硫酸盐硫转移酶）和两个小鼠抗体片段作为模型目标蛋白。当它们在大肠杆菌中单独表达时，会形成不溶性聚集体。它们的基因被设计通过一个含有凝血酶切割位点的寡肽连接到TcFKBP18的C末端，从而作为融合蛋白表达。通过融合TcFKBP18，目标蛋白在可溶部分的表达显著增加。表达蛋白中可溶形式的百分比达到宿主可溶蛋白的10%-28%。对表达的抗体片段-TcFKBP18融合蛋白进行纯化和蛋白酶消化后，切割后的抗体片段（单链Fv）在ELISA中显示出与抗原的特异性结合。这表明表达的抗体片段正确折叠成了活性形式。
