PURIFICATION, CHARACTERIZATION, AND GENETIC ORGANIZATION OF RECOMBINANT PROVIDENCIA-STUARTII UREASE EXPRESSED BY ESCHERICHIA-COLI
PURIFICATION, CHARACTERIZATION, AND GENETIC ORGANIZATION OF RECOMBINANT PROVIDENCIA-STUARTII UREASE EXPRESSED BY ESCHERICHIA-COLI
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DOI：
10.1128/jb.170.5.2202-2207.1988
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发表时间：
1988-05-01
影响因子：
3.2
通讯作者：
HAUSINGER, RP
中科院分区：
文献类型：
--
作者：
MULROONEY, SB;LYNCH, MJ;HAUSINGER, RP
来自斯氏普罗维登斯菌的重组脲酶已在大肠杆菌中表达并纯化，并且结构基因的遗传组织已确定。脲酶表达由尿素诱导并被培养基中的富氮成分抑制。脲酶蛋白经 DEAE-Sepharose、苯基-Sepharose、Mono-Q 和苯基-Superose 色谱纯化 331 倍，产率 7.3%。该酶对尿素的Km为9.3mM，并且以7,100μmol/min/mg的Vmax水解尿素。 P. Stuartii 脲酶由三种多肽（Mrs、73,000、10,0000 和 9,000）组成，分别表示为 α、β 和 γ。基于通过凝胶过滤色谱法获得的天然 Mr 230,000 和考马斯亮蓝，天然酶最好描述为 (α.1.beta.2.gamma.2)2各个亚基的染色强度。纯蛋白质的原子吸收分析显示1.9.+-。每 alpha.1.beta.2.gamma.2 单位含有 0.1 个镍离子。重组脲酶的转座子插入突变体的体外转录-翻译分析证明，脲酶肽在相邻DNA序列上编码，并以γ、β、然后α的顺序转录为多顺反子mRNA。鉴定出三种脲酶缺陷型插入突变体，其不影响脲酶亚基多肽的合成，这表明一些镍加工酶激活或其他功能对于产生活性脲酶也可能是必要的。
Recombinant urease from Providencia stuartii has been expressed in and purified from Escherichia coli, and the genetic organization of the structural genes has been determined. Urease expression was induced by urea and repressed by nitrogen-rich components in the medium. The urease protein was purified 331-fold by DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and phenyl-Superose chromatographies with a 7.3% yield. The enzyme possessed a Km for urea of 9.3 mM and hydrolyzed urea at a Vmax of 7,100 .mu.mol/min per mg. P. stuartii urease is composed of three polypeptides (Mrs, 73,000, 10,0000, and 9,000) denoted by .alpha., .beta., and .gamma.. The native enzyme is best described as (.alpha.1.beta.2.gamma.2)2, based on a native Mr of 230,000, obtained by gel filtration chromatography, and on the Coomassie blue staining intensities of the individual subunits. Atomic absorption analysis of the pure protein revealed 1.9 .+-. 0.1 nickel ions per .alpha.1.beta.2.gamma.2 unit. In vitro transcription-translation analysis of transposon insertion mutants of the recombinant urease demonstrated that the urease peptides are encoded on adjacent DNA sequences and transcribed as a polycistronic mRNA in the order of .gamma., .beta., and then .alpha.. Three urease-defective insertion mutants were identified that did not affect synthesis of urease subunit polypeptides, indicating that some nickel processing enzyme activation, or other function may also be necessary for producing an active urease.