Expression and high yield production of the catalytic domain of matrix metalloproteinase 12 and of an active mutant with increased solubility
Expression and high yield production of the catalytic domain of matrix metalloproteinase 12 and of an active mutant with increased solubility
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DOI：
10.1016/s1381-1169(03)00493-x
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发表时间：
2003-09-15
影响因子：
--
通讯作者：
Terni, B
中科院分区：
文献类型：
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作者：
Banci, L;Bertini, I;Terni, B
在一个旨在筛选几种不同基质金属蛋白酶（MMP）候选药物以寻找选择性原理的项目的总体框架中，MMP-12（金属弹性酶）的催化结构域已在大肠杆菌菌株BL21D3中表达，其产量优化到约30 mg/dm(3)。所选择的结构跨越整个MMP-12的106-267残基，并在104-105位包含两个额外的蛋氨酸。这与先前发表的跨度残基99-279的结构不同[J]。中国生物医学工程学报，2001,12 (2):444 - 444 [J]。生物学杂志，312(2001)7311。该蛋白在包涵体中表达，在高浓度尿素中溶解，并在温和抑制剂乙酰羟肟酸的存在下，通过逐步尿素稀释，从1 H到N-15 HSQC判断，适当地重新折叠。当需要进行活性或抑制研究时，后者可以很容易地透析出来。这种催化结构域结构的MMP-12的溶解度约为250-300 muM。为了增加溶解度，设计了一种突变体（F171D），该突变体不应改变活性，也不应干扰全长蛋白中催化结构域与前后的前结构域或c端结构域之间的接触。F171D突变体获得了完全活性，可溶性是WT的3倍，极大地促进了其在核磁共振筛选实验中的应用。目前x射线资料的比较[j]。化学。Int。编辑42 (2003)2673][J]。Mol. Biol. 312 (2001) 731；J. Mol. Biol. 312 (2001) 743] MMP-12催化结构域的结构表明，消除Met 105和延伸264-267应该导致更可溶性和更稳定的结构。后者被生产出来，并显示出所需的性能。(C) 2003 Elsevier B.V.版权所有
In the general frame of a project aiming at screening candidate drugs against several different matrix metalloproteinases (MMP) to find a rationale for selectivity, the catalytic domain of MMP-12 (metalloelastase) has been expressed in E. coli strain BL21D3 and its production optimized to about 30 mg/dm(3). The chosen construct spans residues 106-267 of the whole MMP-12 and contains two additional methionines at positions 104-105. This is at variance with the previously published constructs which span residues 99-279 [J. Mol. Biol. 312 (2001) 743] and 100-262 [J. Mol. Biol. 312 (2001) 7311, respectively. The protein, expressed in inclusion bodies, is solubilized in high urea concentration and properly refolded, as judged from 1 H to N-15 HSQC, by stepwise urea dilution in the presence of the mild inhibitor acetohydroxamic acid. The latter can be easily dialysed out when needed for activity or inhibition studies. The solubility of this catalytic domain construct of MMP-12 is around 250-300 muM. To increase solubility, a mutant (F171D) has been designed that should not alter the activity and should not interfere with the contacts between the catalytic domain and either the pro-domain or the C-terminal domains that precede and follow it in the full-length protein. The F171D mutant was produced and indeed resulted fully active and three times more soluble than the WT, greatly facilitating its use in NMR screening experiments. Comparison of X-ray data for the present [Angew. Chem. Int. Ed. 42 (2003) 2673] and previous [J. Mol. Biol. 312 (2001) 73 1; J. Mol. Biol. 312 (2001) 743] constructs of the catalytic domain of MMP-12 suggested that elimination of Met 105 and of the stretch 264-267 should lead to an even more soluble and stable construct. The latter was produced and showed to have the desired properties. (C) 2003 Elsevier B.V. All rights reserved.