| Expression and Purification of Recombinant Skd3 (Human ClpB) Protein and Tobacco Etch Virus (TEV) Protease from Escherichia coli
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Author:
Date:
2020-12-05
[Abstract] Skd3 (encoded by human CLPB) is a mitochondrial AAA+ protein comprised of an N-terminal ankyrin-repeat domain and a C-terminal HCLR-clade nucleotide-binding domain. The function of Skd3 has long remained unknown due to challenges in purifying the protein to high quality and near homogeneity. Recently we described Skd3 as a human mitochondrial protein disaggregase that solubilizes proteins in the mitochondrial intermembrane space. This protocol overcomes the challenges associated with purifying Skd3 and allows for in depth in vitro study of Skd3 activity. Tobacco etch virus (TEV) protease is required in the purification of Skd3. Thus, we also describe how to purify high quality TEV protease for use in the purification of Skd3, other purification protocols, and in vitro assays requiring TEV ...
[摘要] [摘要] Skd3（由人类CLPB编码）是一种线粒体AAA +蛋白，由N末端锚蛋白重复域和C末端HCLR分支核苷酸结合域组成。由于在纯化蛋白质达到高质量和接近均质性方面的挑战，Skd3的功能长期未知。最近，我们描述Skd3作为人类线粒体蛋白disaggregase ，在线粒体膜间间隙增溶蛋白。该协议克服了与纯化Skd3相关的挑战，并允许对Skd3活性进行深入的体外研究。Skd3的纯化需要烟草蚀刻病毒（TEV）蛋白酶。因此，我们还描述了如何净化高质量TEV蛋白酶可用于纯化Skd3，其他纯化方案以及需要TEV蛋白酶的体外测定。
[背景] Skd3是一种线粒体AAA +蛋白，与多系统线粒体疾病VII型3-甲基谷氨酸酸尿症（MGCA7）有关（Capo-Chichi等人，2015； Kanabus等人，2015； Saunders等人，， 2015; Wortmann等人，2015 ; Kiykim等人，2016 ）。由于在体外研究Skd3的能力有限，因此对生物学功能和这些突变对Skd3活性的影响的研究仍难以捉摸（Cupo和Shorter，2020； ...
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| Rapid Isolation and Purification of Secreted Bacteriocins from Streptococcus mutans and Other Lactic Acid Bacteria
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Author:
Date:
2020-11-20
[Abstract] Bacteriocins are small ribosomally synthesized antimicrobial peptides produced by some microorganisms including lactic acid bacteria (LAB), a group of Gram-positive bacteria (cocci, rods) expressing high tolerance for low pH. Bacteriocins kill bacteria rapidly and are biologically active at very low concentrations. Bacteriocins produced by LAB are primarily active against closely related bacterial species. Many bacteriocins have been investigated with respect to their potential use in promoting human, plant, and animal health, and as food biopreservatives. Bacteriocins produced by LAB are particularly interesting since several LAB have been granted GRAS (Generally Recognized as Safe) status. Because it is not always possible to extract active bacteriocins secreted from cells grown in ...
[摘要] [摘要]细菌素是由一些核糖体合成的抗菌肽微生物，包括乳酸菌（LAB），一组革兰氏阳性菌（球菌，棒）表现出对低pH的高耐受性。细菌素可迅速杀死细菌，并在极高的温度下具有生物活性。低浓度。LAB生产的细菌素主要对紧密相关的细菌具有活性种类。已经研究了许多细菌素在促进细菌生长方面的潜在用途。对人类，植物和动物健康，以及作为食品生物防腐剂。LAB生产的细菌素是特别有趣的是，由于一些实验室已获得GRAS（通常被认为是安全的）状态。因为并非总是可能提取液体中生长的细胞分泌的活性细菌素介质，我们开发了一种使用半固体的简单而廉价的肽提取程序营养丰富的琼脂培养基。我们在此提出一种详细的程序，以快速提取出从口腔物种分泌的生物活性细菌素肽变异链球菌，多产的菌种的生产及其在其他实验室提取细菌素的潜在应用（例如，链球菌，乳球菌，肠球菌）。我们还提出了一种简单的检测方法纯化的细胞外肽提取物的细菌素活性测定
[背景]自然界中的大多数细菌并不是独立存在，而是存在于复杂的多物种中生物膜群落（López等，2010）。两者之间存在大量的身体和营养相互作用细菌有助于生物膜的生长和存活。细菌素的产生和分泌细胞外空间为生产者与其他竞争者赋予了独特的生态优势存在于同一生态位中的细菌（Donia and ...
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