Optimized production and purification of Bacillus anthracis lethal factor
Optimized production and purification of Bacillus anthracis lethal factor
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DOI：
10.1006/prep.2000.1208
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发表时间：
2000-04-01
影响因子：
1.6
通讯作者：
Leppla, SH
中科院分区：
文献类型：
--
作者：
Park, S;Leppla, SH
研究了对数正态粒径分布的矩方法在气溶胶凝聚问题中的精度。常碰撞核凝聚问题是通过矩量法以及非常精确的数值方法（Landgrebe和Pratsinis，1990）来解决的。通过不同的力矩选择，得到了问题的近似解析解，并与精确数值模型的结果进行了比较。在分析过程中，解决方案的时刻的尺寸分布得到了精确的形式，它被讨论的解决方案可以作为一个标准的参考，以确保任何新的数值格式求解凝聚方程的精度。通过用对数正态尺寸分布函数表示尺寸分布来获得粒度分布的时间演化。根据分析过程中三个时刻的选择，获得了不同的尺寸分布参数的解决方案。作为例子，比较了三个时刻的选择。使用0（第），1（第）和2（第）的时刻，这是传统上使用的被证明是最准确的各种多分散性的情况下，通过比较数值结果，并建议用于未来的study. Bacillusanthracis致死因子（LF）是一个90 kDa的锌金属蛋白酶，在生物体的毒力中起着重要作用。LF以前曾从大肠杆菌和炭疽杆菌中提纯过，这些制备物的产率和纯度不足以进行晶体结构测定。在这项研究中，将编码野生型LF和突变的失活LF（LF-E687 C）的基因置于E.大肠杆菌-芽孢杆菌穿梭载体，使LF产生的保护性抗原（PA）的信号肽在其N-末端。得到的载体pSJ 115和pSJ 121分别表达野生型和突变的LF融合蛋白。LF基因的表达受PA启动子的控制，在分泌过程中，PA信号肽被切割以释放90-kDa LF蛋白。使用三个色谱步骤（苯基-琼脂糖、Q-琼脂糖和羟基磷灰石）从培养基中纯化野生型和突变的LF蛋白。纯化的蛋白质纯度大于95%，产量（20-30 mg/L）高于其他表达系统中获得的产量（1-5 mg/L）。这些蛋白质已经结晶，并被用于解决LF的晶体结构。还讨论了它们在炭疽疫苗中的潜在用途。(C)北京大学出版社.
The accuracy of the moment method of log-normal size distribution for aerosol coagulation problem was investigated. The constant collision kernel coagulation problem was solved by the moment method as well as a very accurate numerical method (Landgrebe and Pratsinis, 1990) for the purpose. Approximate analytical solutions to the problem by different choices of moments were obtained and compared with the result of the accurate numerical model. During the analysis, solutions to the moments of the size distribution were obtained in exact form and it was discussed that the solutions can be used as a standard reference for ensuring the accuracy of any new numerical schemes for solving the coagulation equation. The time evolution of the particle size distribution was obtained by representing the size distribution with a log-normal size distribution function. Depending on the choice for three moments during the analysis, different solutions to the size distribution parameters were obtained. Three choices for moments were compared as an example. The use of the 0(th), 1(st) and 2(nd) moments which has been conventionally used was proven to be most accurate for various polydispersity cases by comparing with a numerical result and is recommended to be used for future studies.Bacillus anthracis lethal factor (LF) is a 90-kDa zinc metalloprotease that plays an important role in the virulence of the organism. LF has previously been purified from Escherichia coli and Bacillus anthracis, The yields and purities of these preparations were inadequate for crystal structure determination. In this study, the genes encoding wild-type LF and a mutated, inactive LF (LF-E687C) were placed in an E. coli-Bacillus shuttle vector so that LF was produced with the protective antigen (PA) signal peptide at its N-terminus. The resulting vectors, pSJ115 and pSJ121, express wild-type and mutated LF fusion proteins, respectively. Expression of the LF genes is under the control of the PA promoter and, during secretion, the PA signal peptide is cleaved to release the 90-kDa LF proteins. The wild-type and mutated LF proteins were purified from the culture medium using three chromatographic steps (Phenyl-Sepharose, Q-Sepharose, and hydroxyapatite). The purified proteins were greater than 95% pure and yields (20-30 mg/L) were higher than those obtained in other expression systems (1-5 mg/L). These proteins have been crystallized and are being used to solve the crystal structure of LF. Their potential use in anthrax vaccines is also discussed. (C) 2000 Academic Press.