蛋白质药物糖基化工程化改造研究进展

多种哺乳和非哺乳动物的蛋白质表达系统已成功用于重组糖蛋白药物的生产。糖基化对于生物药品的研究开发至关重要,对生物药品的药效、半衰期及抗原性等产生重要影响。糖基化工程的目的是生产组分明晰、结构均一的N-和O-连接的糖基化蛋白药物。N-糖基化改造的相关研究显示,利用哺乳动物和非哺乳动物表达系统可以表达均匀的N-聚糖重组糖蛋白。与N-糖基化改造相比, O-糖基化的改造研究尚处于起步阶段。首个糖基化工程单克隆抗体已在美国和日本获得上市批准。综述了重组蛋白表达系统的糖基化工程化改造的研究进展,包括蛋白质药物的 N-糖基化改造和O-糖基化改造的最新进展,以期为蛋白质药物的糖基化工程改造研究提供参考。     

植物激素糖基化修饰研究进展

植物激素对植物的生长发育有重要的调节作用。由于激素的作用依赖于其浓度, 所以植物内源活性激素的水平必须受到严格控制, 而糖基化修饰被认为是调控激素活性水平的重要方式之一。随着植物激素糖基化修饰相关糖基转移酶基因不断被克隆与鉴定, 多种植物激素的糖基化修饰机制和功能作用逐渐被揭示。该文重点介绍了近年来植物生长素、细胞分裂素、脱落酸、油菜素内酯、水杨酸、茉莉酸等植物激素的糖基转移酶活性鉴定与功能研究进展。同时, 对植物激素糖基化修饰领域存在的问题和发展前景进行了讨论。     

酵母N-糖基化工程研究进展

酵母表达系统可用来生产具生物活性的重组糖蛋白,但其在N-糖基化过程中会生成高甘露糖型糖链。通过引入相关的甘露糖苷酶和糖基转移酶基因、切断酵母自身的高甘露糖链形成通道能够改变酵母宿主N-糖基化的类型。本对酵母N-糖基化工程的研究状况、最新进展及存在问题作简要阐述。     

糖基转移酶在去糖基化酶

在糖基化工程中,通过酶法对蛋白质进行糖基化和修饰和对天然糖蛋白去糖基化是研究糖蛋白结构与功能的重要手段。本文综述了近年来所纯化的主要的糖基化转移酶和去糖基化酶的性质和应用。     

糖基转移酶和去糖基化酶

在糖基化工程中,通过酶法对蛋白质进行糖基化修饰和对天然糖蛋白去糖基化是研究糖蛋白结构与功能的重要手段。本文综述了近年来所纯化的主要的糖基化转移酶和去糖基化酶的性质和应用。     

植物小分子化合物的糖基化与糖基转移酶

文章介绍近年来植物体内亲脂性小分子化合物的糖基化和与其相关的糖基转移酶研究进展.     

天然产物的C-糖基化研究进展

自然界中的天然产物在其结构上存在各种修饰,C-糖基化为其中一种比较稀少的修饰方式.C-C糖苷键的形成由C-糖基转移酶负责催化.含C-糖基的化合物多数来自微生物,但高等植物也会有少量积累.综述了近些年来在天然产物C-糖基化方面的研究工作,并对其在药物开发方面的潜力进行了展望.     

植物糖基转移酶及其在代谢工程中的应用

糖基转移酶催化植物次生代谢物合成,在植物的生长发育过程以及代谢工程应用方面起着重要作用。该文介绍糖基转移酶的基本特性,总结近年来研究植物糖基转移酶基因的克隆与功能分析的方法,详细阐述了微生物中3个C-糖基转移酶基因（UrdGT2、gilGT和iroA）的克隆和功能研究,概括糖基转移酶在代谢工程的研究进展,并展望今后的研究趋势,为植物C-糖基转移酶的生物学功能研究和代谢应用方面提供有益帮助。     

分子伴侣协助下抗肿瘤抗生素美达霉素生物合成中的糖基转移酶Med-ORF8 的原核表达

糖基转移酶在天然产物的糖基化修饰过程中起关键作用。目前链霉菌源抗肿瘤抗生素美达霉素(Medermycin)生物合成途径中的糖基转移酶Med-ORF8的原核表达及酶学性质还未有研究。首先通过结构模拟确定Med-ORF8的端部加上His-标签不会影响其三维结构的正确折叠,然后利用2种pET原核表达载体来进行Med-ORF8的原核表达,发现以pET-28a(+)为载体进行表达时,目的蛋白产量非常高,但是以不可溶的包涵体形式为主。当分子伴侣基因(编码大肠杆菌触发因子)与Med-ORF8的编码基因共表达时,在优化诱导条件的情况下,可以有效减少包涵体的形成,提高了Med-ORF8的可溶性表达效率,为Med-ORF8的酶学分析打下基础。     

天然产物的糖基化及糖基侧链改造

抗生素和抗癌药物等多种天然产物的活性都依赖于其糖基侧链,糖基侧链结构的变化对母体化合物的生物活性、底物适应性及药理学性质具有重要影响。糖基侧链结构变化多端,修饰、改变天然产物的糖基侧链已成为获得临床候选药物的重要方法。利用化学法和酶法,研究者创造了多种改造天然产物糖基化的方法。详细介绍了天然产物的糖基化过程,并从组合生物学、糖基转移酶改造、糖类随机化及新型糖类随机化和糖基转移酶可逆性四方面阐述了糖基侧链的改造方法。     

Immobilization of galactosyltransferase and continuous galactosylation of glycoproteins in a reactor

We immobilized human milk galactosyltransferase covalently to CNBr- and tresylchloride-activated Sepharose. The enzyme was also immobilized non-covalently to Concanavalin A-Sepharose and to monoclonal anti-galactosyltransferase antibodies which were boundvia their Fc-fragment to Protein G-Sepharose. With the covalent methods, up to 72% of the enzyme could be bound to the carrier, but more than 90% of the specific activity was lost. In contrast, non-covalent immobilization yielded only about 50% immobilization efficiency, but 21% and 25% of specific activity, respectively, could be recovered. The stability of immobilized galactosyltransferase as compared to native enzyme was considerably increased: at room temperature, 55% of initial immobilized activity was lost after 65 hours compared to 95% of loss of soluble enzyme activity. Immobilized galactosyltransferase was then used for continuous galactosylation of the glycoproteins ovalbumin, endo H-treated yeast invertase and bovine serum albumin-N-acetylglucosamine in a slurry reactor. 55%, 35% and 25%, respectively, of all acceptor sites on these glycoproteins could be galactosylated by this method.     

Glycosylation of flavonoids with a glycosyltransferase from Bacillus cereus

Microbial glycosyltransferases can convert many small lipophilic compounds such as phenolics, terpenoids, cyanohydrins and alkaloids into glycons using uridine-diphosphate-activated sugars. The main chemical functions of glycosylation processes are stabilization, detoxification and solubilization of the substrates. The gene encoding the UDP-glycosyltransferase from Bacillus cereus, BcGT-1, was cloned by PCR and sequenced. BcGT-1 was expressed in Escherichia coli BL21 (DE3) with a his-tag and purified using a His-tag affinity column. BcGT-1 could use apigenin, genistein, kaempferol, luteolin, naringenin and quercetin as substrates and gave two reaction products. The enzyme preferentially glycosylated at the 3-hydroxyl group, but it could transfer a glucose group onto the 7-hydroxyl group when the 3-hydroxyl group was not available. The reaction products made by biotransformation of flavonoids with E. coli expressing BcGT-1 are similar to those produced with the purified recombinant enzyme. Thus, this work provides a method that might be useful for the biosynthesis of flavonoid glucosides and for the glycosylation of related compounds.     

抗克伦特罗单链抗体表达载体的构建与鉴定

为构建克伦特罗(Clenbuterol, CBL)单链抗体(scFv)表达载体, 实现其在大肠杆菌中的表达。以pCANTAB5E- CBL质粒为模板, 扩增CBL的scFv基因, 与pPICZaA载体重组, 然后以重组质粒pPICZaA-scFv为模板扩增scFv及其相连的6 个组氨酸(6×His)片段, 再与载体pBV220连接, 转化大肠杆菌DH5a, 阳性克隆质粒经酶切和PCR鉴定后进行目的片断的序列测定。重组菌经温度诱导表达重组单链抗体, 并通过SDS-PAGE和Western blotting对其鉴定。采用竞争ELISA检测重组单链抗体的抗原结合活性。结果表明重组质粒pBV220-scFv含有插入片段, 与原序列的同源性达99.8%。重组蛋白的分子量接近37 kD, 能够被抗His标签单克隆抗体特异性识别且可被游离的CBL竞争性抑制, IC50值为4.55 ng/mL。这说明我们成功构建了重组质粒pBV220-scFv并实现了其在大肠杆菌中的表达, 为进一步进行CBL免疫法快速检测奠定了一定的基础。     

Expression of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase isoforms in murine tissues determined by real-time PCR: a new view of a large family

The members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family transfer GalNAc to serine and threonine sites and initiate mucin-type O-glycosylation. There are at least 13 functionally characterized family members in mammals. Explanations for the large size of this enzyme family have included functional redundancy, differences among isoforms in substrate specificity, and specific expression of individual isoforms in particular tissues or during certain developmental stages. To date no quantitative comparison of the levels of all ppGaNTase isoforms in any tissue of any species has been reported. We performed real-time polymerase chain reaction using the Taqman method to determine the expression of ppGaNTase isoforms in mouse tissues. Several tissues exhibited a common pattern in which isoforms T1 and T2 were the most strongly expressed, although the level of expression varied widely among tissues. In striking contrast to this general pattern, testis, sublingual gland, and colon exhibited distinctive profiles of isoform expression. Isoform T13 was expressed most strongly in brain, and one putative isoform was expressed only in testis. In mammary tissue the expression of several isoforms changed markedly during pregnancy and lactation. In summary these real-time PCR data indicate the contribution of each isoform to the overall ppGaNTase expression within each tissue and highlight the particular isoforms and tissues that will be the targets of future studies on the functions of the ppGaNTase family.     

植物UGTs的功能、应用与分子改造研究进展

植物尿苷二磷酸糖基转移酶（uridinediphosphate,UGTs）,在次生代谢产物生物合成、激素平衡、免疫防御、脱毒反应等生理过程中发挥重要作用。随着酶功能表征和结构研究的不断深入,具有催化高效性、区域/立体选择性和底物宽容性的植物UGTs可用于天然/非天然糖苷的酶法直接合成及异源生物合成,植物UGTs酶分子理性改造与定向进化的成功例子也在不断增加。围绕生物学功能、糖基化应用与酶分子改造三个方面,对近几年来植物UGTs的相关研究进行系统综述。同时,对植物UGTs基因的准确预测与筛选、系统功能分析及酶分子理性设计等方面的发展趋势进行展望。     

重组人半乳凝集素-1的原核表达、纯化及生物活性检测

目的:在大肠杆菌中表达半乳凝集素-1(galectin-1),并进行纯化及生物活性检测。方法:将人半乳凝集素-1基因克隆至带有His融合标签的原核表达载体pQE-30上,转化大肠杆菌M15,经IPTG诱导表达,表达产物经亲和层析纯化后,进行Western印迹鉴定,并用红细胞凝集试验检测其生物学活性。结果:双酶切鉴定和核苷酸序列测定表明重组表达质粒pQE-30-Galectin-1构建正确;重组蛋白的表达量约占菌体总蛋白的50%,主要以可溶形式表达,纯化后蛋白纯度达95%以上,且具有良好的红细胞凝集活性。结论:在大肠杆菌中表达了重组人半乳凝集素-1,且具有良好的生物活性。     

The Formation of Sugar Chains in Triterpenoid Saponins and Glycoalkaloids

Triterpenoid saponins and structurally related steroidal glycoalkaloids are a large and diverse family of plant glycosides. The importance of these compounds for chemical protection of plants against microbial pathogens and/or herbivores is now well-documented. Moreover, these compounds have a variety of commercial applications, e.g. as drugs or raw materials for pharmaceutical industry. Until recently there were only sparse data on the biosynthesis of saponins and glycoalkaloids, especially at the enzyme level. Substantial progress has recently been made, however, in our understanding of biosynthetic routes leading to the formation of the diverse array of aglycone skeletons found in these compounds as well as mechanisms of synthesis of their sugar moieties. This review highlights some of the advances made over past two decades in our understanding of the formation and modification of sugar moieties in triterpenoid saponins and glycoalkaloids.     

Glycosyltransferases:key players involved in the modification of plant secondary metabolites

Glycosyltransferases are members of the multigene superfamily in plants that can transfer single or multiple activated sugars to a range of plant molecules,resulting in the glycosylation of plant compounds.Although the activities of many glycosyltransferases and their products have been recognized for a long time,only in recent years were some glycosyltransferase genes identified and a few functionally characterized in detail.Glycosylation is thought to be one of the most important modification reactions towards plant secondary metabolites,and plays a key role in maintaining cell homeostasis,thus likely participating in the regulation of plant growth,development and in defense responses to stress environments.With advances in plant genome projects and the development of novel technologies in analyzing gene function,significant progress could be made in gaining new insights into the properties and precise biological roles of plant secondary product glycosyltransferases,and the new knowledge will have extensive application prospects in the catalytic synthesis of glycoconjugates and metabolic engineering of crops.In this review,we summarize the current research,highlighting the possible biological roles,of plant secondary metabolite glycosyltransferases and discuss their potential applications as well as aspects to be further studied in the near future.     

Glycosyltransferases: key players involved in the modification of plant secondary metabolites

Glycosyltransferases are members of the multigene superfamily in plants that can transfer single or multiple activated sugars to a range of plant molecules, resulting in the glycosylation of plant compounds. Although the activities of many glycosyltransferases and their products have been recognized for a long time, only in recent years were some glycosyltransferase genes identified and a few functionally characterized in detail. Glycosylation is thought to be one of the most important modification reactions towards plant secondary metabolites, and plays a key role in maintaining cell homeostasis, thus likely participating in the regulation of plant growth, development and in defense responses to stress environments. With advances in plant genome projects and the development of novel technologies in analyzing gene function, significant progress could be made in gaining new insights into the properties and precise biological roles of plant secondary product glycosyltransferases, and the new knowledge will have extensive application prospects in the catalytic synthesis of glycoconjugates and metabolic engineering of crops. In this review, we summarize the current research, highlighting the possible biological roles, of plant secondary metabolite glycosyltransferases and discuss their potential applications as well as aspects to be further studied in the near future.