Chevalier N, Callens M, Michels P A
Research Unit for Tropical Diseases, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Protein Expr Purif. 1995 Feb;6(1):39-44. doi: 10.1006/prep.1995.1006.
A procedure has been developed for high-level expression of Trypanosoma brucei fructose-bisphosphate aldolase in Escherichia coli. Therefore, a specific restriction site was introduced by mutagenesis at the front of the gene, enabling its ligation in an expression plasmid, immediately downstream of the regulatory sequences. Growth conditions were established for production of high amounts of soluble and active enzyme. Aldolase was purified to near-homogeneity from the soluble fraction of the bacterial lysate by nuclease treatment, differential precipitation steps, and passage over a CM-Sepharose column. From a 1-liter culture of E. coli cells, 60-120 mg of purified protein that is essentially indistinguishable in physicochemical and kinetic properties and in stability from the enzyme purified from trypanosomes grown in infected laboratory animals was reproducibly obtained.
已开发出一种在大肠杆菌中高效表达布氏锥虫果糖二磷酸醛缩酶的方法。因此，通过诱变在基因前端引入了一个特定的限制性酶切位点，使其能够连接到表达质粒中，紧接在调控序列下游。建立了用于大量生产可溶性活性酶的生长条件。通过核酸酶处理、分步差速沉淀以及在CM-琼脂糖柱上进行层析，从细菌裂解物的可溶部分将醛缩酶纯化至近乎均一。从1升大肠杆菌细胞培养物中，可重复性地获得60 - 120毫克纯化蛋白，该蛋白在物理化学性质、动力学性质以及稳定性方面与从感染实验动物体内生长的锥虫中纯化得到的酶基本无差异。
