Heterologous expression and purification of Vibrio proteolyticus (Aeromonas proteolytica) aminopeptidase: a rapid protocol.
Heterologous expression and purification of Vibrio proteolyticus (Aeromonas proteolytica) aminopeptidase: a rapid protocol.
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DOI：
10.1016/j.pep.2009.02.011
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发表时间：
2009-07
影响因子：
1.6
通讯作者：
Bennett, Brian
中科院分区：
文献类型：
--
作者：
Hartley, Mariam;Bennett, Brian
金属氨基肽酶(MAP)是参与HIV感染性、肿瘤生长和转移、血管生成和细菌感染的酶。研究MAP的构效关系是合理设计抗MAP化疗药物的前提。在生物医学上重要的双核图谱中，研究最多的成员是典型的分泌型蛋白溶解弧菌双锌氨基肽酶(VPAP)。野生型酶很容易从蛋白溶解弧菌的培养上清液中纯化，但重组变异体需要在大肠杆菌中表达。本文介绍了一种大大改进的重组VPAP纯化系统。用金属离子亲和层析法从大肠杆菌培养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。这一步骤取代了以前方法中的(NH4)2SO4分级、阴离子交换和疏水作用层析分离的顺序。传统上，重组VPAP酶原用蛋白酶K和加热(70℃)处理，去除N-末端和C-末端区域，得到成熟的活性酶。这种方法不适用于对这些治疗不稳定的VPAP变种。在新方法中，利用了VPAP自催化N-末端前肽和C-末端区域的能力，对高纯度的VPAP-(His)6全长多肽进行了广泛的透析，获得了成熟的活性蛋白，而不需要依赖于蛋白酶K或热处理。因此，对以前未被分离为成熟形式的蛋白质的变体进行了纯化。
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 (NH4)2SO4 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 °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.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
2.9
作者：
EDELHOCH, H
通讯作者：
EDELHOCH, H
影响因子：
2.9
作者：
ARFIN, SM;BRADSHAW, RA
通讯作者：
BRADSHAW, RA
影响因子：
3
作者：
Babst, R;Bongiorno, L;Urbani, A
通讯作者：
Urbani, A
影响因子：
5.3
作者：
BLONDIN, J;TAYLOR, A
通讯作者：
TAYLOR, A