Belzer C A, Tabatabai L B, Frank G H
Center for Veterinary Biologics Licensing and Policy Development Ames, IA 50010, USA.
Prep Biochem Biotechnol. 2000 Nov;30(4):343-55. doi: 10.1080/10826060008544973.
Pasteurella haemolytica serovar A1 is the causative agent of acute fibrinohemorrhagic pneumonia also known as shipping fever. Many pathogens, including P. haemolytica, survive in their respective hosts through the up-regulation of an iron acquisition system. In this study we identified, purified and characterized a 35-kDa periplasmic iron-regulated protein. The N-terminal sequence of the iron-regulated protein ANEVNVYSYRQP YLIEPMLK was identical to the deduced amino acid sequence of the ferric binding protein, FbpA, of P. haemolytica. Growth of P. haemolytica in a synthetic medium (RPMI-1640), without iron and supplemented with 50 gM 2,2' dipyridyl, facilitated the expression, isolation and purification of the native P. haemolytica FbpA. The protein was purified to homogeneity by using ammonium sulfate precipitation followed by CM-Sepharose ion exchange chromatography. SDS-PAGE showed a single band with a molecular weight of 35,369. Isoelectric focusing showed multiple bands with pIs of 5.5, 5.6, 5.8, and one major band with pI of 6.4. The molecular weight obtained by electrospray mass spectrometry was 35,822. Equilibrium velocity ultracentrifugation established that the protein existed as a monomer under native conditions with an apparent molecular weight of 33,795. Analysis of secondary structure of FbpA by circular dichroism showed 42.1% alpha helical structure. This protein is the second periplasmic iron-regulated protein described for P. haemolytica.
溶血巴斯德氏菌A1血清型是急性纤维蛋白出血性肺炎（也称为运输热）的病原体。包括溶血巴斯德氏菌在内的许多病原体通过上调铁摄取系统在各自的宿主中存活。在本研究中，我们鉴定、纯化并表征了一种35 kDa的周质铁调节蛋白。铁调节蛋白的N端序列ANE VNVYSYRQP YLIEPMLK与溶血巴斯德氏菌铁结合蛋白FbpA的推导氨基酸序列相同。溶血巴斯德氏菌在不含铁并添加50 μM 2,2'-联吡啶的合成培养基（RPMI-1640）中生长，有助于天然溶血巴斯德氏菌FbpA的表达、分离和纯化。通过硫酸铵沉淀，然后进行CM-琼脂糖离子交换色谱，将该蛋白纯化至同质。SDS-PAGE显示一条分子量为35,369的单带。等电聚焦显示多个pI为5.5、5.6、5.8的条带，以及一个pI为6.4的主要条带。电喷雾质谱法测得的分子量为35,822。平衡速度超速离心表明，该蛋白在天然条件下以单体形式存在，表观分子量为33,795。通过圆二色性对FbpA二级结构的分析显示，其α螺旋结构占42.1%。该蛋白是为溶血巴斯德氏菌描述的第二种周质铁调节蛋白。
