猪乳铁蛋白在4种重组乳杆菌中的表达及抑菌活性比较

为了获得优化的猪乳铁蛋白乳杆菌表达系统,并比较重组猪乳铁蛋白的抑菌活性,根据乳杆菌使用密码子的偏嗜性优化合成猪乳铁蛋白成熟肽编码序列,将其克隆到乳杆菌表达载体pPG612.1的XhoⅠ/BamHⅠ位点,获得了plf乳杆菌表达载体质粒pPG612.1-plf。将获得的重组质粒分别电转化入干酪乳杆菌ATCC393、戊糖乳杆菌KLDS1.0413、植物乳杆菌KLDS1.0344和副干酪乳杆菌KLDS1.0652细胞内,获得4种表达猪乳铁蛋白的重组乳杆菌。经木糖诱导,通过Western blotting和激光共聚焦检测重组猪乳铁蛋白的表达,用ELISA方法检测和比较4种重组菌上清中表达猪乳铁蛋白的量,并用琼脂孔穴扩散抑菌法检测4种重组乳杆菌表达乳铁蛋白的抑菌活性。结果表明,乳铁蛋白在4种重组乳杆菌中均得到正确表达,其产物分子量约73 kDa,重组干酪乳杆菌、重组戊糖乳杆菌、重组植物乳杆菌和重组副干酪乳杆菌的重组猪乳铁蛋白表达量分别为9.6μg/mL、10.8μg/mL、12.5μg/mL、9.9μg/mL。重组猪乳铁蛋白对大肠杆菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、巴氏杆菌和李氏杆菌均有一定的抑菌作用,对金黄色葡萄球菌的抑菌作用最强,且4种重组乳杆菌中重组植物乳杆菌表达产物的抑菌效果优于其他重组菌的表达产物。结果表明在4种乳杆菌中重组猪乳铁蛋白的最佳表达系统为植物乳杆菌,该结果为猪乳铁蛋白的乳杆菌表达系统进一步开发与应用奠定了基础。     

重组猪乳铁蛋白N端的高效表达及抑菌活性检测

为获得表达猪乳铁蛋白基因的重组菌株,并检测其表达的重组猪乳铁蛋白抑菌活性,应用RT-PCR方法从泌乳3d后母猪乳腺组织中扩增了猪乳铁蛋白N端1077bp的PLF-N基因片段,与GenBank上发表的4株猪乳铁蛋白基因序列相比,核苷酸同源性均达到99%以上。为了得到高表达量的PLF-N基因,以扩增的PLF-N片段为参考模板,经过密码子优化,全基因合成了编码猪乳铁蛋白N端的基因PLF-NS。将其定向插入到原核表达载体pET-30b中,转化大肠杆菌BL21,获得了表达PLF-NS的重组菌pET-PLF-NS/BL21;经IPTG诱导,并对表达条件进行优化,以及通过SDS-PAGE和Western blotting分析均表明猪乳铁蛋白得到了正确表达,其产物分子量约为42kDa,最优表达条件下蛋白表达量占菌体总蛋白的32%,表达产物以包涵体形式存在。包涵体经裂解、纯化、复性处理后纯度达到98%。用琼脂孔穴扩散抑菌法检测表明重组猪乳铁蛋白具有明显的抑菌作用。表明通过基因优化对表达量低的基因进行改造使之高效表达,是一种提高表达效率的有效手段。     

乳蛋白的主要组分及其研究现状

乳蛋白是乳中最重要的成分,包括酪蛋白、乳清蛋白和乳脂肪球膜蛋白等。本文对乳中主要蛋白质的结构组成特点、分泌的规律和功能等进行了综述,并介绍了国内外乳蛋白研究的最新进展及其研究乳蛋白的意义。     

原料乳冷藏过程中微生物细胞的宏蛋白组学分析

原料乳在投入生产前通常需4°C冷藏,在这个过程中微生物的污染将造成冷藏原料乳的变质。【目的】本文旨在分子水平上探究原料乳冷藏过程中微生物表达的差异蛋白质的动态变化,为原料乳的冷藏提供理论支撑。【方法】利用Label-free技术研究原料乳4°C冷藏6 d期间微生物菌体蛋白质的物种来源、功能及参与通路,筛选差异蛋白质并对主要差异蛋白质参与的通路进行富集,探究其变化规律。【结果】冷藏过程中共鉴定出341个微生物菌体蛋白质,其中冷藏3 d后产生的蛋白质占所有检出蛋白质的60.12%。COG及KEGG分析表明,蛋白质所体现的功能及参与的通路随时间而变化。冷藏4 d,参与糖酵解/糖异生、ABC转运蛋白、氨基糖和核苷酸糖代谢等通路的蛋白数量显著增加。随冷藏时间延长,相邻时间点差异蛋白质数目逐渐增多,且富集在不同的通路中。【结论】冷藏过程中原料乳中的微生物所产生的蛋白质参与的通路及其所体现的功能复杂,4 d时的变化最为明显,或可作为原料乳质量控制的关键节点。     

基于iTRAQ技术对植物乳杆菌FS5-5的耐盐特性分析

【目的】对大酱中耐盐性较好的植物乳杆菌进行蛋白质组学研究,为植物乳杆菌盐胁迫应激机制的研究提供实验数据。【方法】本项研究以筛选自东北传统农家大酱的耐盐性较好的植物乳杆菌FS5-5为研究对象,绘制了其在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下的生长曲线,并利用i TRAQ技术研究了其在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下的蛋白质表达情况。【结果】植物乳杆菌FS5-5在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下到达对数生长期中期的时间点分别为5、10、12和12 h;以差异倍数在1.2倍以上且P0.05为筛选条件对6.0%、7.0%和8.0%(W/V)Na Cl浓度下与0%进行差异蛋白质的筛选,共筛选出1271个差异蛋白质。这些差异蛋白质主要参与糖代谢、氨基酸代谢、脂肪酸代谢、核苷酸代谢、应激反应、转运、PTS系统和核糖体代谢等。【结论】植物乳杆菌在高盐浓度下生长与能量合成蛋白质、应激蛋白质以及相容性溶质转运蛋白质的表达上调有密切关系。     

猪肠道乳杆菌的筛选及其生物学特性研究

目的从健康仔猪肠道中筛选到用于研制微生态制剂的优良乳杆菌株。方法本研究选用乳杆菌选择培养基LBS、MRS从健康仔猪肠道分离到67株乳杆菌,对其染色镜检、生化试验和耐酸、耐胆汁、耐高温、抑菌活性及动物安全性等生物学特性进行初步研究。结果从其中筛选到3株嗜酸乳杆菌,均能耐受pH3．0的酸度、0．8％～1．0％的牛胆盐和60℃的高温,其代谢产物对猪肠道致病性大肠埃希菌和猪沙门菌具有抑制作用,且对小白鼠无致病性。结论3株嗜酸乳杆菌符合益生菌的要求,可作为猪用益生素制剂的候选菌株。     

巴马小型猪肝硬化前后肠道乳杆菌的比较研究

目的比较巴马小型猪诱导型肝硬化造模前(正常猪)和造模成功后(肝硬化猪)肠道乳杆菌的变化情况,探讨小型猪肝硬化模型在肝硬化肠道微生态研究中的适用性。方法收集肝硬化造模前和CCl4诱导肝硬化造模成功后巴马小型猪的新鲜粪便,提取粪便总菌DNA,用乳杆菌特异性引物进行PCR扩增,对扩增产物进行变性梯度凝胶电泳(Denaturing Gradient Gel-Electrophoresis,DGGE),即用PCR-DGGE分析巴马小型猪肝硬化前后肠道乳杆菌的相似性和多样性。结果聚类分析和主成分分析显示巴马小型猪肝硬化前(正常猪)和肝硬化后混杂排列,无明显界限;多样性数据分析显示巴马小型猪肝硬化前后肠道乳杆菌的丰富度(S)、微生物区系Shannon-Wiener指数(H′)和均匀度(E)差异均无统计学意义(P0.05)。结论巴马小型猪肝硬化后肠道乳杆菌与正常猪比较差异无统计学意义。     

乳蛋白质水解液脱苦方法研究

通过对乳蛋白质酶水解液的脱苦研究,结果表明:乳蛋白质在酶水解过程中,随着水解度的增加,苦味值增大;在水解度为66％时,苦味值最大;随后苦味值减小;3％粉末状活性碳对水解液的脱苦效果好。     

猪乳中一组高分子量蛋白的多态性与乳生长因子、窝增重的关系

利用十二烷基硫酸钠-聚丙烯酰胺凝胶不连续垂直板电泳(SDS-PAGE),对162头二花脸母猪乳中一组高分子量蛋白质(HMWP)进行了检测和分型,并运用线型模型统计分析方法分析了该基因座的不同基因型与母猪的乳生长因子(IGF-1、EGF和胰岛素)、哺乳仔猪生长(20日龄窝增重)的关系.结果表明,在三种HMWP基因型中,不同基因型母猪的乳中IGF-1浓度存在显著差异,HMWP基因型为BB型和BD型的母猪,其乳IGF-1浓度均高于DD型,其中BB型显著高于DD型(P＜0.05).乳中胰岛素浓度也存在差异的趋势,但未达到显著水平(P＞0.05),BB型和BD型母猪的乳中胰岛素浓度高于DD型.HMWP基因座不同基因型的乳EGF浓度无显著差异(P＞0.05).在三种HMWP基因型中,不同基因型母猪的20日龄窝增重存在显著差异,HMWP基因座为BB型和BD型的母猪,其20日龄窝增重均高于DD型,其中BD型显著高于DD型(P＜0.05).实验结果提示,HMWP多态性可能作为一个潜在的遗传标记应用于猪的遗传育种.     

猪生长瘦肉和沉积脂肪的分子与生化基础

我国畜牧业中一直存在着有碍生产、不利遗传、又难以解释的问题。亦即中国地方猪瘦肉少脂肪多 ,如湖北通城猪和江苏太湖猪 ;而外来大型猪则相反 ,瘦肉多脂肪少。对于这种复杂问题 ,华中农业大学生命科学技术学院杨在清等教授和博士们作了研究 ,发现通城猪和太湖猪血液中激素敏感脂酶活性很高 ,还有一种分子量为 4 47KDa的细胞质蛋白质和分子量为 94KDa的脂细胞膜蛋白质。这些都是外来猪斯格猪和长白猪所极少有的 ,特殊的和带有遗传性的体内物质。这些物质经蛋白质SDS PAGE分析发现 ,敏感脂酶活性比外来猪高 3倍 ,4 47KDa蛋白质和 94KD…     

Isolation and identification of a high molecular weight protein in sow milk

A high molecular weight protein (HMWP) was isolated and purified from sow milk, and some of its biochemical characteristics and biological functions were identified. The origin of HMWP was also investigated. The molecular weight of HMWP was determined to be about 115 000 and 114 800 by SDS-PAGE and gel filtration, respectively. The sequence of 10 amino acids in N-terminal of HMWP was Ala-Leu-Val-Gln-Ser-Cys-Leu-Asn-Leu-Val. The sequence was blasted against GenBank. No protein showed significant similarity with this sequence suggesting the HMWP may be novel. The result of liquid chromatography mass spectrometry (LC-MS) also proved HMWP could be a novel protein. By amino acid assay, HMWP was rich in glutamate (including glutamine), cysteine, glycine, aspartic acid (including asparagines) and proline. The content of hydrophobic amino acids (Ala, Val, Leu, Ile, Met, Phe and Pro) was lower at 18.59% of the total amino acids suggesting HMWP has high solubility in water. Western blots of lectins were used to identify the kinds of carbohydrate residues attached to HMWP qualitatively. The result showed that HMWP was a kind of glycoprotein containing N-acetylneuraminic acid (NeuNAc), mannose (Man) and/or N-acetylglucosamine (GlcNAc). By isoelectric focusing, HMWP pI was found to be 5.1. Compared with milk fat globule membrane protein (MFGMP) isolated from the sow milk in SDS-PAGE, MFGMP did not contain HMWP. HMWP was assumed to be a secretory milk protein. HMWP was not found in bovine, goat, rabbit or human milk in SDS-PAGE gel suggesting HMWP may be unique to sow milk. By Western blot, HMWP could be detected in sow milk, not in sow serum, which suggests it is synthesized and secreted by the mammary gland. HMWP concentrations in sows milk were the lowest in the first day of lactation, rose significantly during lactation 1 to 7 days. The HMWP content of sows milk remained relatively constant ((1.95±0.13) g/l) during lactation 7 to 20 days. HMWP significantly inhibited Escherichia coli in a dose related manner in vitro. Overall, HMWP could be a novel sow milk protein with implications for the mammary gland and the piglet.     

猪乳中一高分子量蛋白质的分离纯化和鉴定

对猪乳中一高分子量蛋白(HMWP)进行了分离纯化,并对其某些生化性质进行了鉴定。猪乳通过去脂得到脱脂乳,再去除酪蛋白得到乳清。对乳清进行硫酸铵分级盐析,猪乳中HMWP在40％饱和度硫酸铵盐析下有最大沉淀。收集40％饱和度硫酸铵盐析沉淀,经过溶解、透析得到HMWP的粗品。通过Mono Q离子交换柱,对其粗品进行两次层析提纯,得到了HMWP纯品,其纯度和得率分别为97．85％和12．31％。多种植物凝集素的Western blotting鉴定表明,HMWP是一个糖基种类较少的糖蛋白,含有Man和GlcNAc。SDS-PAGE和凝胶过滤分别测得HMWP的分子量为114．8kD和115．0kD。通过等电点测定,HMWP的pI为5．10。HMWP的氨基酸组分分析得知,其富含Asp、Glu、Gly和Cys,疏水性氨基酸较低,仅占15．59％摩尔分数。这些结果说明HMWP是一个易溶于水的、酸性的分泌性单体球蛋白。N端氨基酸序列测定结果为Ala-Leu-Val—Gln-Ser-Gty-Leu-Ash-Leu-Val,通过从网络Genbank检索没有发现其同源蛋白的序列,说明其可能是一个新蛋白。     

Influence of the metabolic state during lactation on milk production in modern sows

Selection for prolificacy in sows has resulted in higher metabolic demands during lactation. In addition, modern sows have an increased genetic merit for leanness. Consequently, sow metabolism during lactation has changed, possibly affecting milk production and litter weight gain. The aim of this study was to investigate the effect of lactational feed intake on milk production and relations between mobilization of body tissues (adipose tissue or skeletal muscle) and milk production in modern sows with a different lactational feed intake. A total of 36 primiparous sows were used, which were either full-fed (6.5 kg/day) or restricted-fed (3.25 kg/day) during the last 2 weeks of a 24-day lactation. Restricted-fed sows had a lower milk fat percentage at weaning and a lower litter weight gain and estimated milk fat and protein production in the last week of lactation. Next, several relations between sow body condition (loss) and milk production variables were identified. Sow BW, loin muscle depth and backfat depth at parturition were positively related to milk fat production in the last week of lactation. In addition, milk fat production was related to the backfat depth loss while milk protein production was related to the loin muscle depth loss during lactation. Backfat depth and loin muscle depth at parturition were positively related to lactational backfat depth loss or muscle depth loss, respectively. Together, results suggest that sows which have more available resources during lactation, either from a higher amount of body tissues at parturition or from an increased feed intake during lactation, direct more energy toward milk production to support a higher litter weight gain. In addition, results show that the type of milk nutrients that sows produce (i.e. milk fat or milk protein) is highly related to the type of body tissues that are mobilized during lactation. Interestingly, relations between sow body condition and milk production were all independent of feed level during lactation. Sow management strategies to increase milk production and litter growth in modern sows may focus on improving sow body condition at the start of lactation or increasing feed intake during lactation.     

High-molecular-weight protein 2 of Yersinia enterocolitica is homologous to AngR of Vibrio anguillarum and belongs to a family of proteins involved in nonribosomal peptide synthesis.

The iron-regulated irp2 gene is specific for the highly pathogenic Yersinia species and encodes high-molecular-weight protein 2 (HMWP2). Despite the established correlation between the presence of HMWP2 and virulence, the role of this protein is still unknown. To gain insight into the function of HMWP2, the entire coding sequence and the promoter of irp2 were sequenced. Two putative -35 and -10 promoter sequences were identified upstream of a large open reading frame, and two potential Fur-binding sites were found overlapping the second -35 box. The large open reading frame is composed of 6,126 nucleotides and may encode a protein of 2,035 amino acids (ca. 228 kDa) with a pI of 5.81. A signal sequence was not present at the N terminus of the protein. Despite the existence of 30 cysteine residues, carboxymethylation prevented the formation of most if not all disulfide bonds that otherwise occurred when the cells were sonicated. The protein was composed of three main domains: a central region of ca. 850 residues, bordered on each side by a repeat of 550 residues. A high degree of identity (44.5%) was found between HMWP2 and the protein AngR of Vibrio anguillarum. The central part of HMWP2 (after removal of a loop of 337 residues) also displayed significant homology with proteins belonging to the superfamily of adenylate-forming enzymes and, like them, possessed a putative ATP-binding motif that is also present in AngR. In addition, HMWP2 shared with the group of antibiotic and enterochelin synthetases a potential amino acid-binding site. Six consensus sequences defining the superfamily and four defining the family of synthetases were derived from the multiple alignment of the 30 sequences of proteins or repeated domains. A phylogenetic tree that was constructed showed that HMWP2 and AngR are in a family composed of Lys2, EntF, and the tyrocidine, gramicidin, and Beta-lactam synthetases. This finding suggests that HMWP2 may participate in the nonribosomal synthesis of small biologically active peptides.     

Tandem heterocyclization activity of the multidomain 230 kDa HMWP2 subunit of Yersinia pestis yersiniabactin synthetase: interaction of the 1-1382 and 1383-2035 fragments.

The six-domain, 2035-amino acid subunit high-molecular weight protein 2 (HMWP2) activates salicylate and two cysteines and loads them covalently on its three carrier protein domains during assembly of the iron-chelating virulence factor, yersiniabactin of the plague bacterium Yersinia pestis. The 1-1382 fragment of HMWP2 (ArCP-Cy1-A), overproduced in Escherichia coli, contains the first three domains: the aryl carrier protein (ArCP) domain, the cysteine specific adenylation domain (A), and the first condensation/cyclization domain (Cy1). The ArCP can be posttranslationally phosphopantetheinylated on Ser52 and then loaded with a salicyl group on the phosphopantetheine (Ppant) thiol by action of the YbtE, a salicyl-AMP ligase. The HMWP2 1-1382 fragment can activate L-cysteine as Cys-AMP. The HMWP2 1383-2035 fragment contains the remaining three domains: two peptidyl carrier proteins (PCP1 and PCP2) separated by a second condensation/cyclization domain (Cy2). Phosphopantetheinylation of the HMWP2 1383-2035 fragment at Ser1439 (PCP1) and Ser1977 (PCP2) facilitates cysteinylation of both thiols by HMWP2 1-1382. When the holo 1-1382 and bis-holo 1383-2035 protein fragments are mixed with ATP, salicylate, and cysteine, four products are slowly released [salicylcysteine (Sal-Cys), (hydroxyphenylthiazolinyl)cysteine (HPT-Cys), HPT-Cys-Cys, and the bisheterocyclic HPTT-Cys], reflecting thiolytic rerouting by cysteine in solution of elongating acyl-S-enzyme intermediates tethered at ArCP, PCP1, and PCP2 carrier protein domains, respectively. Conducting the in trans reconstitution with the S1439A mutant of HMWP2 1383-2035 releases only Sal-Cys, while the S1977A mutant leads to HPT-Cys formation but not HPT-Cys-Cys or HPTT-Cys. These results suggest localization of particular acyl-S-enzyme intermediates to each of the three carrier protein regions and also establish the sequential action of Cy1 and Cy2, with the latter producing the tandem 4,2-bisheterocyclic hydroxyphenylthiazolinylthiazolinyl (HPTT) moiety characteristic of this class of siderophores.     

Purification of a HeLa cell high molecular weight action binding protein and its identification in HeLa cell plasma membrane ghosts and intact HeLa cells

The high molecular weight protein (HMWP) which was previously observed to be a major component of the actin based gels formed by incubating cytoplasmic extracts of HeLa cells at 25 degrees C [Weihing, R. R. (1977) J. Cell Biol. 75, 95-103] has now been purified by gel filtration of 0.6 M KCl extracts of precipitated gels. A few hundred micrograms of HMWP, which is about 90% pure, can be isolated from 4 X 10(9) cells. HMWP can gel muscle actin and cross-link it into filament bundles. Its subunit molecular weight is 250 0000, its Stokes radius is 125 A, and its sedimentation coefficient is 9 S. A native molecular weight of 480 000 was calculated by using the latter two parameters, and therefore the native molecule is a dimer. Its amino acid analysis is nearly indistinguishable from that of macrophage actin binding protein and of mammalian and avian filamins. All of these findings indicate that HMWP is homologous to the latter proteins. However, HeLa cell HMWP and avian filamin must differ in their primary sequences because their partial peptide maps are distinct and because an antiserum against HMWP reacts only weakly with filamin. For studies on the intracellular location of HMWP, a goat antiserum against purified HMWP was prepared and characterized and then used to localize HMWP in suspension grown cells. The technique of immunoblotting revealed that the antiserum reacted virtually exclusively with the high molecular weight polypeptide that comigrates with HMWP in cell lysates and in ZnCl2-stabilized plasma membrane ghosts prepared from HeLa cells [Gruenstein, E., Rich, A., & Weihing, R. R. (1975) J. Cell Biol. 64, 223-234] and that it did not react with rabbit myosin heavy chain, microtubule proteins (MAPS and tubulin) from HeLa cells and calf brain, or the proteins of human erythrocyte ghosts including spectrin. Suspension-grown cells which were stained with the antiserum by the technique of indirect immunofluorescence showed bright fluorescence at the rim of the cells and less intense generalized fluorescence. If preimmune serum or immune serum treated with HMWP was substituted for the immune serum, then staining at the rim was not observed, but the generalized fluorescence was only slightly reduced; unpermeabilized cells were not stained. These results indicate that HMWP is a component of the cortical cytoplasm of HeLa cells. Possible functions of cortical HMWP are discussed briefly.     

Isolation and characterization of a high molecular weight actin-binding protein from Physarum polycephalum plasmodia

A high molecular weight actin-binding protein was isolated from the Physarum polycephalum plasmodia. The protein ( HMWP ) shares many properties with other high molecular weight actin-binding proteins such as spectrin, actin-binding protein from macrophages, and filamin. It has a potent activity to cross-link F-actin into a gel-like structure. Its cross-linking activity does not depend on calcium concentrations. Hydrodynamic studies have revealed that the protein is in the monomeric state of a polypeptide chain with molecular weight of approximately 230,000 in a high ionic strength solvent, while it self-associates into a dimer under physiological ionic conditions. Electron microscopic examinations of HMWP have shown that the monomer particle observed in a high ionic strength solvent is rod shaped with the two-stranded morphology very similar to that of spectrin. On the other hand, under physiological ionic conditions, the HMWP dimer shows the dumb-bell shape with two globular domains connected with a thin flexible strand.     

Yersinia spp. HMWP2, a cytosolic protein with a cryptic internal signal sequence which can promote alkaline phosphatase export.

The iron starvation-induced, 2,042-amino-acid protein HMWP2 of Yersinia enterocolitica has two internal hydrophobic segments which might promote its export and association with the cytoplasmic membrane. To determine whether part of HMWP2 could be exported beyond the periplasmic face of the cytoplasmic membrane, we used TnphoA mutagenesis to construct 10 hybrid proteins in which periplasmic alkaline phosphatase (PhoA) was fused to the end of C-terminally truncated HMWP1 (at amino acid positions 1751 and 1753 two independent isolates]) had high alkaline phosphate activity (close to that of the native enzyme), both in Escherichia coli and in Y. pseudotuberculosis, indicating that the PhoA segment of the hybrid reached the periplasm. Deletion studies showed that the export signal resides in the second hydrophobic segment of HMWP2. This result would be compatible with the topology of the protein in the cytoplasmic membrane predicted from the distribution of charged amino acids at either end of the two hydrophobic segments. However, two hybrids in which the junction was even further toward the C terminus of HMMWP2 (at positions 1793 and 1999) had only weak alkaline phosphatase activity, suggesting that the predicted topology is incorrect. The location of HMWP2 was therefore determined by subcellular fractionation. The results indicate that HMPW2 is mainly cytoplasmic, consistent with its presumed role in the ATP-dependent, nonribosomal synthesis of an unknown peptide. We propose that the high alkaline phosphatase activity associated with some of the HMWP-2-PhoA hybrids results from the unmasking of the cryptic export signal activity in the second hydrophobic segment of HMPW2.     

High-molecular-weight protein (pUL48) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable

We show here that the high-molecular-weight protein (HMWP or pUL48; 253 kDa) of human cytomegalovirus (HCMV) is a functionally competent deubiquitinating protease (DUB). By using a suicide substrate probe specific for ubiquitin-binding cysteine proteases (DUB probe) to screen lysates of HCMV-infected cells, we found just one infected-cell-specific DUB. Characteristics of this protein, including its large size, expression at late times of infection, presence in extracellular virus particles, and reactivity with an antiserum to the HMWP, identified it as the HMWP. This was confirmed by constructing mutant viruses with substitutions in two of the putative active-site residues, Cys24Ile and His162Ala. HMWP with these mutations either failed to bind the DUB probe (C24I) or had significantly reduced reactivity with it (H162A). More compellingly, the deubiquitinating activity detected in wild-type virus particles was completely abolished in both the C24I and H162A mutants, thereby directly linking HMWP with deubiquitinating enzyme activity. Mutations in these active-site residues were not lethal to virus replication but slowed production of infectious virus relative to wild type and mutations of other conserved residues. Initial studies, by electron microscopy, of cells infected with the mutants revealed no obvious differences at late times of replication in the general appearance of the cells or in the distribution, relative numbers, or appearance of virus particles in the cytoplasm or nucleus.