Hartley Mariam, Yong Wei, Bennett Brian
Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226-0509, USA.
Protein Expr Purif. 2009 Jul;66(1):91-101. doi: 10.1016/j.pep.2009.02.011. Epub 2009 Feb 20.
Metalloaminopeptidases (mAPs) are enzymes that are involved in HIV infectivity, tumor growth and metastasis, angiogenesis, and bacterial infection. Investigation of structure-function relationships in mAPs is a prerequisite to rational design of anti-mAP chemotherapeutics. The most intensively studied member of the biomedically important dinuclear mAPs is the prototypical secreted Vibrio proteolyticus di-zinc aminopeptidase (VpAP). The wild-type enzyme is readily purified from the supernatant of cultures of V. proteolyticus, but recombinant variants require expression in Escherichia coli. A greatly improved system for the purification of recombinant VpAP is described. A VpAP-(His)(6) polypeptide, containing an N-terminal propeptide, and a C-terminal (His)(6) adduct, was purified by metal ion affinity chromatography from the supernatant of cultures of E. coli. This single step replaced the sequence of (NH(4))(2)SO(4) fractionation, and anion-exchange and hydrophobic interaction chromatographic separations of earlier methods. Traditionally, recombinant VpAP proenzyme has been treated with proteinase K and with heat (70 degrees C), to remove the N- and C-terminal regions, and yield the mature active enzyme. This method is unsuitable for VpAP variants that are unstable towards these treatments. In the new method, the hitherto noted, but not fully appreciated, ability of VpAP to autocatalyze the hydrolysis of the N-terminal propeptide and C-terminal regions was exploited; extensive dialysis of the highly purified VpAP-(His)(6) full-length polypeptide yielded the mature active protein without recourse to proteinase K or heat treatment. Purification of variants that have previously defied isolation as mature forms of the protein was thus carried out.
金属氨肽酶（mAPs）是一类参与HIV感染、肿瘤生长与转移、血管生成以及细菌感染的酶。研究mAPs的结构-功能关系是合理设计抗mAP化学治疗药物的前提。在具有重要生物医学意义的双核mAPs中，研究最为深入的成员是典型的分泌型溶藻弧菌双锌氨肽酶（VpAP）。野生型酶很容易从溶藻弧菌培养物的上清液中纯化出来，但重组变体需要在大肠杆菌中表达。本文描述了一种大大改进的重组VpAP纯化系统。通过金属离子亲和色谱从大肠杆菌培养物的上清液中纯化出一种含有N端前肽和C端（His）6加合物的VpAP-（His）6多肽。这一步骤取代了早期方法中的硫酸铵分级分离以及阴离子交换和疏水相互作用色谱分离步骤。传统上，重组VpAP酶原一直用蛋白酶K和加热（70℃）处理，以去除N端和C端区域，从而产生成熟的活性酶。这种方法不适用于对这些处理不稳定的VpAP变体。在新方法中，利用了VpAP迄今已被注意到但尚未得到充分认识的自催化水解N端前肽和C端区域的能力；对高度纯化的VpAP-（His）6全长多肽进行广泛透析，无需使用蛋白酶K或热处理即可产生成熟的活性蛋白。从而实现了以前难以分离为成熟形式蛋白质的变体的纯化。
