Siritapetawee Jaruwan, Prinz Heino, Krittanai Chartchai, Suginta Wipa
School of Biochemistry, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
Biochem J. 2004 Dec 15;384(Pt 3):609-17. doi: 10.1042/BJ20041102.
In the present paper, we describe cloning and expression of two outer membrane proteins, BpsOmp38 (from Burkholderia pseudomallei) and BthOmp38 (from Burkholderia thailandensis) lacking signal peptide sequences, using the pET23d(+) expression vector and Escherichia coli host strain Origami(DE3). The 38 kDa proteins, expressed as insoluble inclusion bodies, were purified, solubilized in 8 M urea, and then subjected to refolding experiments. As seen on SDS/PAGE, the 38 kDa band completely migrated to approximately 110 kDa when the purified monomeric proteins were refolded in a buffer system containing 10% (w/v) Zwittergent 3-14, together with a subsequent heating to 95 degrees C for 5 min. CD spectroscopy revealed that the 110 kDa proteins contained a predominant beta-sheet structure, which corresponded completely to the structure of the Omp38 proteins isolated from B. pseudomallei and B. thailandensis. Immunoblot analysis using anti-BpsOmp38 polyclonal antibodies and peptide mass analysis by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS confirmed that the expressed proteins were BpsOmp38 and BthOmp38. The anti-BpsOmp38 antibodies considerably exhibited the inhibitory effects on the permeation of small sugars through the Omp38-reconstituted liposomes. A linear relation between relative permeability rates and M(r) of neutral sugars and charged antibiotics suggested strongly that the in vitro re-assembled Omp38 functioned fully as a diffusion porin.
在本论文中，我们描述了使用pET23d(+)表达载体和大肠杆菌宿主菌株Origami(DE3)克隆和表达两种缺乏信号肽序列的外膜蛋白，即来自类鼻疽伯克霍尔德菌的BpsOmp38和来自泰国伯克霍尔德菌的BthOmp38。以不溶性包涵体形式表达的38 kDa蛋白经纯化后，溶解于8 M尿素中，然后进行复性实验。如SDS/PAGE所示，当纯化的单体蛋白在含有10%（w/v）两性离子去污剂3-14的缓冲系统中复性，并随后在95℃加热5分钟时，38 kDa条带完全迁移至约110 kDa。圆二色光谱显示，110 kDa蛋白含有主要的β-折叠结构，这与从类鼻疽伯克霍尔德菌和泰国伯克霍尔德菌中分离出的Omp38蛋白结构完全一致。使用抗BpsOmp38多克隆抗体进行的免疫印迹分析以及通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）进行的肽质量分析证实，表达的蛋白为BpsOmp38和BthOmp38。抗BpsOmp38抗体对小糖通过Omp38重组脂质体的渗透具有显著的抑制作用。中性糖和带电荷抗生素的相对渗透率与相对分子质量之间的线性关系强烈表明，体外重新组装的Omp38完全发挥了扩散孔蛋白的功能。
