High-cell density shake-flask expression and rapid purification of the large fragment of Thermus aquaticus DNA polymerase I using a new chemically and temperature inducible expression plasmid in Escherichia coli
High-cell density shake-flask expression and rapid purification of the large fragment of Thermus aquaticus DNA polymerase I using a new chemically and temperature inducible expression plasmid in Escherichia coli
复制标题
DOI：
10.1016/j.pep.2008.09.018
复制
发表时间：
2009-02-01
影响因子：
1.6
通讯作者：
Johnson, Kenneth A.
中科院分区：
文献类型：
--
作者：
Brandis, John W.;Johnson, Kenneth A.
我们已经开发了一种新的表达载体pcl（ts）ind（+），该载体基于λ噬菌体的强有力的启动子，该启动子由相同质粒上的λ阻遏物的温度敏感和化学诱导形式控制。将阻遏基因定位在质粒上使得该载体"便携"，因为它可用于转化任何大肠杆菌菌株。因此，可以使用对菌株、诱导条件和收获时间的控制来优化异源蛋白质的产率。为了提供一个概念的证明，我们表明，E。用pcl（ts）ind（+）modKlenTaq1（水生栖热菌DNA聚合酶1的大片段的修饰形式）转化的大肠杆菌recA（+）和recA（-）宿主细胞可以在多个摇瓶中生长至高细胞密度。modKlenTaq1（V649C）的突变版本可以通过简单地将恒温器设置从30 ℃提高到37 ℃并（在recA（+）细胞的情况下）添加萘啶酸以实现完全诱导（总细胞蛋白的12 - 13%）来诱导。使用快速的两步纯化方法，可以从6个2.8升的挡板底摇瓶中纯化近300 mg modKlenTaq1 V649 C，每个摇瓶中容纳1.5 L培养物，最终产量为约33 mg/升或3 mg纯化酶/克细胞湿重。(C)2008年爱思唯尔公司All rights reserved.
We have developed a new expression vector, pcl(ts) ind(+), based upon the powerful rightward promoter of bacteriophage lambda, which is controlled by a temperature-sensitive and chemically-inducible version of the lambda repressor on the same plasmid. Locating the repressor gene on the plasmid makes this vector "portable" in that it can be used to transform any strain of Escherichia coli. Hence, control over strains, induction conditions, and harvest times can be used to optimize yields of heterologous proteins. To provide a proof of concept, we show that E. coli recA(+) and recA(-) host cells transformed with pcl(ts) ind(+) modKlenTaq1 (a modified version of the large fragment of Thermus aquaticus DNA polymerase 1) could be grown to high cell densities in multiple shake-flasks. A mutant version of modKlenTaq1 (V649C) could be induced by simply raising the thermostat setting from 30 to 37 degrees C and (in the case of recA(+) cells) adding nalidixic acid to achieve full induction (12-13% of the total cellular protein). Using a rapid, two-step purification process, it was possible to purify nearly 300 mg of modKlenTaq1 V649C from six 2.8-liter baffle-bottomed shake-flasks each holding 1.5 L of culture for a final yield of approximately 33 mg per liter or 3 mg of purified enzyme per gram of cells wet weight. (C) 2008 Elsevier Inc. All rights reserved.