VIRAL PROTEINASES OF HERPES SIMPLEX TYPE 1 AND COXSACKIEVIRUS, RABBIT PGM AND

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们看到的是兔子和人类的生殖器切割。这种酶是我们核磁共振研究中最大的蛋白质，对细胞中的能量产生至关重要。我们还对病毒蛋白酶感兴趣，例如人类疱疹病毒，它会导致各种严重的疾病。我们有兴趣研究疱疹病毒新的二聚丝氨酸蛋白酶(它具有催化三联体，Ser，His，His，而不是Ser，His，Asp)，它们是病毒成熟所必需的。这项工作的目的是为了更好地了解这些新型蛋白酶的催化机理，这项工作可能会导致新的抑制剂的开发。我们特别感兴趣的是组氨酸在催化部位的作用。我们克隆并高效表达了单纯疱疹病毒1型(HSV-1)和卡波西肉瘤相关病毒(KSHV或8型单纯疱疹病毒，HSV-8)的丝氨酸蛋白酶。疱疹病毒蛋白酶含有18个脯氨酸和几个半胱氨酸，因此需要用复性技术从包涵体中复性蛋白。其他研究小组发表的研究表明，这些蛋白质的产量很低，例如2到5毫克/升，这对我们的需求来说太低了。因此，我不得不改进表达和复性/纯化策略，这些酶具有自催化活性，因此我们有必要开发改进的复性和蛋白质纯化策略，尽快回收尽可能多的活性酶。我已经从小病毒家族柯萨奇病毒B3中纯化了3C半胱氨酸蛋白酶。我也克隆了，过表达了2A蛋白水解酶。柯萨奇病毒与许多严重的人类疾病有关，如心脏病(心肌炎)、呼吸道感染和流感样疾病。其中，2A和3C为单体，分别为16 kDa和20 kDa。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are looking at rabbit and human PGM. This enzyme is the largest protein we are studying by NMR, and it is vital for energy production in cells.We are also interested in viral proteinases, for example human herpesviruses; which cause a wide variety of serious diseases. We are interested in studying the herpesvirus novel dimeric serine proteinases (which possess the catalytic triad, Ser, His, His instead of Ser, His, Asp) that are essential for viral matuartion. The goal of this work is to gain a better understanding of the catalytic mechanism of these novel proteinase, and this work could lead to the development of new inhibitors. We are particularly interested in the role of the histidines at the catalytic site. We have cloned and overexpressed serine proteinases from herpes simplex type-1 virus (HSV-1) and Kaposi sarcoma associated virus (KSHV or herpes virrus type 8 ie., HSV-8).The herpesvirus proteinases contain 18 prolines and several cysteines and therefo re require refolding techniques to renature the protein from inclusion bodies. Published work by other groups show that the yields of these proteins are low e.g. 2 to 5mg/liter, this is too low for our needs. So I have had to improve the expression and refolding/purification strategies Also, these proteinases possess autocatalytic activity, therefore it was essential for us to develop improved refolding and protein purification strategies, that recover as much active enzyme, as quickly as possible.I have purifed 3C cysteine proteinase from Coxsackievirus B3 of the Picornavirus family. I have also cloned, overexpressed 2A proteinase too. Coxsackieviruses have been implicated in many serious human diseases such as heart disease (myocarditis), respiratory infections and flu-like illnesses. The 2A and 3C proteinases are monomeric and 16 kDa and 20 kDa, respectively.