蛋白质剪切及其应用

蛋白质剪切是一种翻译后修饰事件 ,它将插入前体蛋白的中间的蛋白质肽段 (Intein ,internalproteinfrag ment)剪切出来 ,并用正常肽键将两侧蛋白质多肽链 (Extein ,flankingproteinfragments)连接起来。在此过程中不需要辅酶或辅助因子的作用 ,仅需四步分子内反应。Intein及其侧翼序列可以通过突变产生高度特异性的自我切割用于蛋白质纯化、蛋白质连接和蛋白质环化反应 ,在蛋白质工程方面有广泛的应用前景。     

蛋白质剪切及其应用

白质剪切是一种翻译后修饰事件,它将插入前体蛋白的中间的蛋白质肽段（Intein, internal protein fragment）剪切出来,并用正常肽键将两侧蛋白质多肽链（Extein, flanking protein fragments）连接起来。在此过程中不需要辅酶或辅助因子的作用,仅需四步分子内反应。Intein及其侧翼序列可以通过突变产生高度特异性的自我切割用于蛋白质纯化、蛋白质连接和蛋白质环化反应,在蛋白质工程方面有广泛的应用前景。     

一个新的受雄激素诱导的胞浆蛋白的组织分布

本文以３２Ｐ－５＇末端标记ＰＳＢＰ－Ｃ３基因启动子的３１ｂｐＤＮＡ片段作探针，用凝胶阻滞分析法检测了大鼠、小鼠、兔和猴各组织以及人病理组织的胞浆中与此ＤＮＡ片段特异结合的新蛋白质（Ｃ３Ｐ４），以期对其功能的探讨提供必要的线索。结果显示：（１）Ｃ３Ｐ４蛋白以不同的含量存在于４种动物的雄性器官中；（２）４种动物的脑组织均含有此蛋白质，其他组织中很难检测到，且其结果因种族而异：（３）雌性动物中此蛋白质的组织分布除性器官外，与雄性动物相同；（４）在所测定的８种人器官的几种病理组织中，初步观察到胃癌中含量较高，乳房的良性和恶性肿瘤中都有Ｃ２Ｐ４蛋白存在，７例前列腺肥大中有３例比较明显。     

豆薯种子中两种蛋白质的分离纯化及其性质研究

豆薯（Ｐａｃｈｙｒｒｈｉｚｕｓｅｒｏｓｕｓ）种子经磷酸盐缓冲液抽提,Ｓ－ＳｅｐｈａｒｏｓｅＦａｓｔＦｌｏｗ柱,ＤＥ－５２纤维素柱和ＳｅｐｈａｄｅｘＧ－７５柱层析,提取出两种高纯度的蛋白成分,命名为ＰａｃｈｙｒｉｎⅠ和Ⅱ．ＳＤＳ－ＰＡＧＥ测得其分子量分别为３３ｋＤ和１４．５ｋＤ,但ＨＰＬＣ分子筛的结果显示ＰａｃｈｙｒｉｎⅡ的分子量为２８ｋＤ,无论在还原条件下,还是在非还原条件下,ＰａｃｈｙｒｉｎⅡ电泳的结果都完全相同,表明该蛋白的亚基不是以二硫键相连。两种蛋白的等电点分别为４．５和６．５．用酸解法测定了它们的氨基酸组成。在无细胞体系中,它们对蛋白合成有较弱的抑制活性,显示它们可能是核糖体失活蛋白（ＲＩＰｓ）家族中的新成员。     

蛋白质向叶绿体的转运

对近年来叶绿体蛋白质前导肽序列、叶绿体被膜中的蛋白质转运器、监护蛋白在蛋白转运过程中的作用、蛋白质导入叶绿体的途径、前体蛋白的加工的研究进展进行了介绍和评述     

萝卜溶菌酶诱导条件的研究

分别以不同浓度（Ａ４５０ｎｍ表示）的高压灭活白色葡萄球菌、大肠杆菌及水杨酸对萝卜溶菌酶进行不同时间的诱导。结果表明,诱导时间２４ｈ效果最好,白色葡萄球菌最适浓度Ａ４５０ｎｍ为０．９４,酶的活力提高了２．３４倍;大肠杆菌最适浓度是Ａ４５０ｎｍ为０．５５,诱导酶活力提高了０．７５倍,以０．２％的水杨酸为诱导物时,第４ｄ和第８ｄ的效果最好,酶活力分别提高了２．４和１．７倍。以酶作用产物进行田间叶面喷施和     

前体及诱导子和发酵条件对烟曲霉TMS-26产紫杉醇发酵体系优化

紫杉醇是一种广谱的抗癌药物,因其具有独特的抗癌机制、良好的抗癌效果和供不应求的市场等特征而备受关注。紫杉醇具有重大经济效益,但产量受到制约,价格极为昂贵,通过内生真菌发酵法生产紫杉醇能在一定程度上缓解其来源困难的问题。在产紫杉醇内生真菌TMS-26发酵液中添加前体物质和诱导子,并通过对接种量、装液量、初始pH和发酵时间等条件进行优化研究。单因素及正交试验表明在PDB培养基中加入苯丙氨酸20mg/L、苯甲酸钠30mg/L、乙酸钠8g/L、甘氨酸15mg/L、CuSO₄ 0.05mg/L、H₂O₂ 6mmol/L、3,5-二硝基水杨酸15mg/L时能有效提高紫杉醇产量,比优化前增产46.64%,达到446.28µg/L,并且发现最适菌株TMS-26的发酵条件为pH7.5、接种量5%、装液量120mL/250mL、发酵时间为10d。     

一个新的RNF6剪切子spg2的克隆和鉴定

环指蛋白是一类含有环指基序的锌指蛋白,它们主要作为毋泛素连接酶,与成泛素结合酶相结合,促进靶蛋白的降解．应用cDNAarray技术,通过对成人和胚胎睾丸进行基因表达谱分析,获得一在成人睾丸中高表达,胚胎睾丸中低表达的环指结构基因RNF6的不同剪切子spg2．它的全长cDNA的可读框为2055个碱基,编码685个氨基酸残基,其羧基端含有一环指结构．NCBIBLAST显示该基因定位于人13号染色体,含有5个外显子．多组织mRNA表达水平研究显示,它在成人睾丸中高表达,胚胎睾丸中低表达．本研究推测spg2可能通过它的环指结构参与人类睾丸的发育．     

IMPACT-TWIN蛋白纯化系统的机制及其应用

IMPACT-TWIN系统是一种新型的蛋白融合表达及纯化系统,已被应用到蛋白质工程的众多领域。此系统中目的蛋白与蛋白自剪接元件intein及几丁质结合蛋白域CBD构成融合蛋白,利用几丁质亲和层析柱,通过诱导内含肽的肽键裂解活性,使目的蛋白释放出来,而内含肽与几丁质结合蛋白仍结合在几丁质介质上,实现了单柱分离纯化蛋白。本文主要阐述了IMPACT-TWIN蛋白表达纯化系统的机制、特点,以及其在蛋白表达纯化方面应用的最新进展。     

基因重组大肠杆菌表达HrpNEcc蛋白的发酵条件及诱导条件优化

对已构建好的表达HrpNEcc蛋白的工程菌BL21(DE3)/pET30a(+)hrpN Ecc的摇瓶发酵条件及乳糖诱导进行优化, 通过在7L发酵罐中放大发酵实验,以期提高蛋白产量并降低生产成本。在摇瓶中优化的发酵及诱导条件是：5% 的接种量,TB培养基,菌体培养至对数生长前期,添加3g/L外源诱导剂乳糖时,HrpNEcc蛋白产量可达417.60mg/L,比不添加乳糖时提高了36.73%,比用IPTG诱导时提高了16.85%。7L发酵罐中发酵,获得菌体湿重达到57.24g/L（WCW）,可溶性HrpNEcc蛋白产量占细胞总蛋白的50.2%,为3.29 g/L。     

蛋白质剪接与一类新的可移动遗传因子

最近在几种生物体中连续发现了蛋白质剪接现象,由于它与通常所说的RNA剪接的区别,人们称这些在蛋白质水平被剪切掉的部分为“蛋白质内含子”.有些蛋白质内含子具有核酸内切酶功能,编码蛋白质内含子的DNA片段是一类新的可移动遗传因子.文中介绍了目前发现的几例蛋白质剪接现象,讨论了蛋白质剪接的可能机理,分析了蛋白质内含子的进化及其生物学意义.     

蛋白质剪接及其在蛋白质工程中的应用

蛋白质剪接是蛋白质内含肽介导的,一种在蛋白质水平上翻译后的加工过程,它由一系列分子内的剪切-连接反应组成。蛋白质内含肽是一个蛋白质前体中的多肽序列,可以催化自身从蛋白质前体中断裂,使两侧的蛋白质外显肽连接成成熟的蛋白质。蛋白质内含肽的发现,不仅丰富了遗传信息翻译后加工的理论,在实践中也有广泛的应用前景。Abstract: Protein splicing , which is an intein mediated posttranslational processing, involves a series of intramolecular cleavage-ligation reactions. Intein is an intervening polypeptide which can catalytic self-cleavage from a pre-protein accompanied by the concomitant joining of the two flanking polypeptides (the extein) through a peptide bond. Protein splicing not only enriches genetic theory of posttranslational processing, but also have wide application prospect.     

A New Method for Purifing Cyt b6f Protein Complex

A new protocol which is much simpler than current procedures, has been developed for purification of the Cyt b6f protein complex. The protocol contained only two steps dialysis-cen- trifugation and stepwise precipitation with ammonium sulfate. Moreover, this method is suitable for larger scale preparation. The purified complex from spinach ( Spinacia oleracea L. ) contained 9.8 nmol Cyt f per milligram protein. 2 Cyt b6(b-hemes) and 1 Chl a per Cyt f. SDS-PAGE showed four main bands and one weak band with low molecular weight. Its activity(PQ2H2→Cyt c)was around 80 μmol Cyt c·nmol Cyt f-1·h-l     

Structural and Dynamical Features of Inteins and Implications on Protein Splicing

Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave themselves from larger precursor proteins and ligate their flanking polypeptides (exteins) through a multistep chemical reaction. First thought to be an anomaly found in only a few organisms, protein splicing by inteins has since been observed in microorganisms from all domains of life. Despite this broad phylogenetic distribution, all inteins share common structural features such as a horseshoe-like pseudo two-fold symmetric fold, several canonical sequence motifs, and similar splicing mechanisms. Intriguingly, the splicing efficiencies and substrate specificity of different inteins vary considerably, reflecting subtle changes in the chemical mechanism of splicing, linked to their local structure and dynamics. As intein chemistry has widespread use in protein chemistry, understanding the structural and dynamical aspects of inteins is crucial for intein engineering and the improvement of intein-based technologies.     

Splicing and alternative splicing in rice and humans

Rice is a monocot gramineous crop, and one of the most important staple foods. Rice is considered a model species for most gramineous crops. Extensive research on rice has provided critical guidance for other crops, such as maize and wheat. In recent years, climate change and exacerbated soil degradation have resulted in a variety of abiotic stresses, such as greenhouse effects, lower temperatures, drought, floods, soil salinization and heavy metal pollution. As such, there is an extremely high demand for additional research, in order to address these negative factors. Studies have shown that the alternative splicing of many genes in rice is affected by stress conditions, suggesting that manipulation of the alternative splicing of specific genes may be an effective approach for rice to adapt to abiotic stress. With the advancement of microarrays, and more recently, next generation sequencing technology, several studies have shown that more than half of the genes in the rice genome undergo alternative splicing. This mini-review summarizes the latest progress in the research of splicing and alternative splicing in rice, compared to splicing in humans. Furthermore, we discuss how additional studies may change the landscape of investigation of rice functional genomics and genetically improved rice. [BMB Reports 2013; 46(9): 439-447]     

Protein Splicing: How Inteins Escape from Precursor Proteins

Inteins are nature''s escape artists; they facilitate their excision from flanking polypeptides (exteins) concomitant with extein ligation to produce a mature host protein. Splicing requires sequential nucleophilic displacement reactions catalyzed by strategies similar to proteases and asparagine lyases. Inteins require precise reaction coordination rather than rapid turnover or tight substrate binding because they are single turnover enzymes with covalently linked substrates. This has allowed inteins to explore alternative mechanisms with different steps or to use different methods for activation and coordination of the steps. Pressing issues include understanding the underlying details of catalysis and how the splicing steps are controlled.