Boesen C C, Motyka S A, Patamawenu A, Sun P D
Structural Biology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852, USA.
Protein Expr Purif. 2000 Oct;20(1):98-104. doi: 10.1006/prep.2000.1306.
Expression systems have been designed to test the suitability of expressing the high cysteine containing extracellular domain (residues 1-136) of human transforming growth factor beta type II receptor (TbetaRII). Receptor expressed using a baculovirus system was functional following both enzymatic deglycosylation and elimination of the N-terminal 22 amino acids by protease degradation. Bacterial expression of a TbetaRII lacking the 26 N-terminal amino acids retained the ability to bind its ligand, TGF-beta1. Receptor expressed in bacteria was sensitive to proteolytic degradation at residue Lys98 but a K98T mutation eliminated degradation and did not disrupt binding. Although several different forms of TbetaRII were expressed, only a fusion with glutathione S-transferase gave soluble TbetaRII, which was purified at a yield of 0.1 mg/10 L of bacterial growth. N-Terminal truncations of TbetaRII (residues 22-136 or 27-136) could be refolded from inclusion bodies and purified to an active form with an efficiency of 10%.
已设计表达系统来测试表达人转化生长因子β II型受体（TbetaRII）含高半胱氨酸的细胞外结构域（第1至136位氨基酸残基）的适用性。使用杆状病毒系统表达的受体在酶促去糖基化以及通过蛋白酶降解去除N端22个氨基酸后仍具有功能。缺少26个N端氨基酸的TbetaRII在细菌中的表达保留了结合其配体TGF-β1的能力。在细菌中表达的受体对第98位赖氨酸残基处的蛋白水解降解敏感，但K98T突变消除了降解且未破坏结合。尽管表达了几种不同形式的TbetaRII，但只有与谷胱甘肽S-转移酶的融合产生了可溶性TbetaRII，其以0.1 mg/10 L细菌生长的产量纯化。TbetaRII的N端截短形式（第22至136位或第27至136位氨基酸残基）可以从包涵体中重折叠并纯化至活性形式，效率为10%。
