Sticha K R, Sieg C A, Bergstrom C P, Hanna P E, Wagner C R
Department of Medicinal Chemistry, University of Minnesota, Minneapolis 55455, USA.
Protein Expr Purif. 1997 Jun;10(1):141-53. doi: 10.1006/prep.1997.0734.
N-Acetyltransferases (NATs) are enzymes that catalyze the detoxification and/or bioactivation of a variety of xenobiotics. Rapid kinetic, biophysical, structural, and bioactivation studies on NATs require quantities of purified enzyme capable of being obtained only through recombinant DNA technology. This laboratory has previously developed a protein expression and purification system in which NATs are expressed as proteins fused to a FLAG octapeptide followed by a thrombin-cleavage site to allow liberation of the rNAT. Typically, however, only 0.5-1.5 mg of the recombinant NAT's could be readily purified in a single isolation sequence by immunoaffinity chromatography. Therefore, the expression system was modified by inserting the L54F dihydrofolate reductase (DHFR) mutant gene sequence between the FLAG octapeptide and the thrombin-cleavage site. Expression was carried out with TOPP3 Escherichia coli cells. The new purification methodology utilizes the unique pH dependence of binding to a methotrexate (MTX)-affinity column by the L54F DHFR mutant. Unfortunately, the affinity chromatography strategy did not work satisfactorily. Although the specific activity of the purified rNAT2 was comparable to that of NAT2 obtained from hamster tissue, only 3% of the activity was recovered. The apparent cause of the low recovery is the unanticipated irreversible binding of rNAT2 to MTX. Ion-exchange chromatography was investigated as an alternative purification method. An initial DEAE anion-exchange column resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin and reapplied to a DEAE anion-exchange column. The second DEAE column resulted in not only the separation of rNAT2-70D from FLAG-L54F DHFR, but also the purification of rNAT2-70D to near homogeneity. Application of the nearly homogeneous rNAT2-70D to a gel-filtration column resulted in recovery of homogeneous protein. The ion-exchange method of purifying rNAT2-70D is inexpensive and simple and yields more than 8 mg of pure enzyme from 1 liter of cell culture.
N - 乙酰转移酶（NATs）是一类催化多种外源性物质解毒和/或生物活化的酶。对NATs进行快速动力学、生物物理、结构及生物活化研究需要大量纯化的酶，而这只能通过重组DNA技术获得。本实验室先前开发了一种蛋白质表达和纯化系统，其中NATs被表达为与FLAG八肽融合的蛋白质，随后是凝血酶切割位点，以便释放重组NAT（rNAT）。然而，通常在单次免疫亲和层析分离过程中，只能轻松纯化出0.5 - 1.5毫克的重组NAT。因此，通过在FLAG八肽和凝血酶切割位点之间插入L54F二氢叶酸还原酶（DHFR）突变基因序列对表达系统进行了修饰。使用TOPP3大肠杆菌细胞进行表达。新的纯化方法利用了L54F DHFR突变体与甲氨蝶呤（MTX）亲和柱结合的独特pH依赖性。不幸的是，亲和层析策略效果并不理想。尽管纯化的rNAT2的比活性与从仓鼠组织获得的NAT2相当，但仅回收了3%的活性。回收率低的明显原因是rNAT2与MTX意外的不可逆结合。研究了离子交换层析作为一种替代纯化方法。最初的DEAE阴离子交换柱实现了融合蛋白的部分纯化。融合蛋白用凝血酶切割后重新应用于DEAE阴离子交换柱。第二个DEAE柱不仅实现了rNAT2 - 70D与FLAG - L54F DHFR的分离，还将rNAT2 - 70D纯化至接近均一性。将几乎均一的rNAT2 - 70D应用于凝胶过滤柱可回收均一的蛋白质。纯化rNAT2 - 70D的离子交换方法廉价且简单，从1升细胞培养物中可获得超过8毫克的纯酶。
