大肠杆菌BL21(DE3)中DnaE intein介导ABCA1蛋白的连接

摘 要：【目的】利用Ssp DnaE intein的蛋白质反式剪接技术研究在大肠杆菌中对ABCA1基因表达产物的连接作用。【方法】将ABCA1的cDNA于满足剪接所需的保守性氨基酸Cys978密码子前断裂为N端和C端两部分,分别与天然存在的反式作用Ssp DnaE intein的123个氨基酸的N端和36个氨基酸的C端编码序列融合,构建到原核表达载体pET-28a(+)。转化感受态大肠杆菌BL21(DE3)细胞,诱导表达后观察重组蛋白的表达和ABCA1的连接。【结果】转化菌经IPTG诱导表达,SDS-PA     

Split Inteins介导的多肽链两端标记

多肽链多位点特异性标记有助于了解蛋白质的结构与功能,特别是在蛋白质的动态构象研究方面. 但是,现有的多肽链多位点特异性标记方法各有局限性,并且种类有限,所以有必要开发新的多肽链多位点特异性标记方法以满足研究需求. 本文以Diub (ubiquitin dimer) 蛋白为研究对象,借助S1和S11型2种不同的断裂蛋白质内含子 (split inteins) 的蛋白质反式剪接,将含有不同荧光基团的两种小肽成功剪接至靶蛋白的两端,最终达到对靶蛋白的末端标记目的.     

Ssp DnaB intein大肠杆菌中介导FVIII重链和轻链的连接

研究利用intein的蛋白质反式剪接功能在大肠杆菌中对凝血VIII因子(FVIII)重链和轻链的连接作用,将B结构域大部分缺失型FVIII(BDD-FVIII)于满足剪接所需的保守性氨基酸Ser1657前断裂为重链和轻链,分别与split mini Ssp DnaB intein的106个氨基酸的N端(Int-N)和48个氨基酸的C端(Int-C)融合,构建到原核表达载体pBV220。诱导表达后SDS-PAGE分析可见预期大小的BDD-FVIII蛋白条带,Western blotting用FVIII特异性抗体证明其为剪接所产生的BDD-FVIII蛋白,表明intein可有效连接BDD-FVIII的重链和轻链。为进一步甲型血友病基因治疗研究应用intein以双腺相关病毒载体(AAV)携带FVIII基因,克服单个AAV载体的容量限制提供了依据。     

Solid phase synthesis of peptide-selenoesters

The synthesis of proteins by native chemical ligation greatly enhances the application of chemistry to complex molecules such as proteins. The essential building blocks for this approach traditionally have been peptide-thioester segments that are linked chemoselectively in consecutive reactions. By using peptide selenoesters instead of thioesters, the ligation rate can be significantly accelerated permitting couplings at difficult sites and potentially enabling new ligation strategies. To facilitate the routine synthesis of selenoester peptides, a general and straightforward procedure has been developed that generates a suitably functionalized resin from which the desired selenoester peptide can be readily synthesized. This simple approach utilizes readily available and cheap chemical agents and enables production of peptide selenoesters of excellent quality in short time and with high recovery. In addition, the stability of peptide selenoesters was examined under different native chemical ligation conditions and compared to thioesters. Selenoesters are slightly more reactive and more susceptible to hydrolysis and aminolysis than thioesters but sufficiently stable under mildly acidic conditions (pH 6.5). Under these conditions, rapid selenoester-mediated ligation is kinetically favoured.     

Total chemical synthesis of the B1 domain of protein L from Peptostreptococcus magnus

Reported here is a native chemical ligation strategy for the total chemical synthesis of the B1 domain of protein L. A synthetic construct of this 76 amino acid protein domain was prepared by the chemoselective ligation of two unprotected polypeptide fragments, one containing an N-terminal cysteine residue and one containing a C-terminal thioester moiety. The polypeptide fragments utilized in the ligation reaction were readily prepared by stepwise solid phase peptide synthesis (SPPS) methods for Boc-chemistry. The milligram quantities of protein required for conventional biophysical studies were readily accessible using the synthetic protocol described here. The folding properties of the synthetic protein L construct were also determined and found to be very similar to those of a similar wild-type protein L constructs prepared by recombinant-DNA methods. This work facilitates future unnatural amino acid mutagenesis experiments on this model protein system to further dissect the molecular basis of its folding and stability.     

Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter were ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.     

Development of a fluorogenic sensor for activated Cdc42

Cdc42, a member of the Rho GTPase family, is a fundamental regulator of the actin cytoskeleton during cell migration. To generate a sensor for Cdc42 activation, we employed a multi-pronged approach, utilizing cysteine labeling and expressed protein ligation, to incorporate the environment sensitive fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) into the GTPase binding domain of the WASP protein. These constructs bind only the active, GTP-bound conformation of Cdc42 to produce a fluorescence signal. Studies with a panel of five sensor analogs revealed a derivative that exhibits a 32-fold increase in fluorescence intensity in the presence of activated Cdc42 compared to incubation with the inactive GDP-bound form of the protein. We demonstrate that this sensor can be exploited to monitor Cdc42 nucleotide exchange and GTPase activity in a continuous, fluorescence assay.     

Consequences of post-translational modifications on amyloid proteins as revealed by protein semisynthesis

Alterations to the global levels of certain types of post-translational modifications (PTMs) are commonly observed in neurodegenerative diseases. The net influence of these PTM changes to the progression of these diseases can be deduced from cellular and animal studies. However, at the molecular level, how one PTM influences a given protein is not uniform and cannot be easily generalized from systemic observations, thus requiring protein-specific interrogations. Given that protein aggregation is a shared pathological hallmark in neurodegeneration, it is important to understand how these PTMs affect the behavior of amyloid-forming proteins. For this purpose, protein semisynthesis techniques, largely via native chemical and expressed protein ligation, have been widely used. These approaches have thus far led to our increased understanding of the site-specific consequences of certain PTMs to amyloidogenic proteins’ endogenous function, their propensity for aggregation, and the structural variations these PTMs induce toward the aggregates formed.     

Efficient and systematic synthesis of a small glycoconjugate library having human complex type oligosaccharides

To prepare a small library of homogeneous glycoconjugates with varying oligosaccharide structures, a combinatorial strategy was employed. The target glycopeptide was divided into two peptide segments (A and B) and both were prepared by solid phase peptide synthesis. These peptides, which can be coupled by native chemical ligation through an amide bond, were subsequently coupled to two kinds of human complex type oligosaccharides. This process systematically afforded the desired glycoconjugate library.     

Synthesis and use of a pseudo-cysteine for native chemical ligation.

The process of native chemical ligation (NCL) is well described in the literature. An N-terminal cysteine-containing peptide reacts with a C-terminal thioester-containing peptide to yield a native amide bond after transesterification and acyl transfer. An N-terminal cysteine is required as both the N-terminal amino function and the sidechain thiol participate in the ligation reaction. In certain circumstances it is desirable, or even imperative, that the N-terminal region of a peptidic reaction partner remain unmodified, for Instance for the retention of biological activity after ligation. This work discusses the synthesis of a pseudo-N-terminal cysteine building block for incorporation into peptides using standard methods of solid phase synthesis. Upon deprotection, this building block affords a de facto N-terminal cysteine positioned on an amino acid sidechain. which is capable of undergoing native chemical ligation with a thioester. The syntheses of several peptides and structures containing this motif are detailed, their reactions discussed. and further applications of this technology proposed.     

TMD2前断裂CFTR翻译后的连接及氯离子通道功能

CFTR基因突变导致一种常染色体隐性遗传疾病——囊性纤维化(CF)。利用split Ssp DnaB intein的蛋白质反式剪接技术的真核细胞双载体转CFTR基因,旨在研究翻译后水平CFTR的连接,以及由其建立的氯离子通道功能。于CFTR膜内第2个跨膜结构域(TMD2)前的Glu838密码子后将其cDNA断裂为N端和C端两部分,与具有蛋白质反式剪接作用的split Ssp DnaB intein编码序列融合,分别插入到载体pEGFP-N1和pEYFP-N1,构建一对真核表达载体pEGFP-NInt和pEYFP-IntC。用脂质体将这对载体共转染至幼年仓鼠肾细胞(BHK),瞬时表达实验用Western blotting观察CFTR蛋白质的连接,并用膜片钳技术记录Cl-通道电流。结果显示,基因共转染细胞呈现完整的CFTR蛋白条带,膜片钳记录到全细胞Cl-电流和单个Cl-通道开放活性。结果表明split Ssp DnaB intein的蛋白质反式剪接技术可用于双载体共转移CFTR基因,为CF基因治疗应用双腺相关病毒载体(AAV)转运CFTR基因,克服AAV的容量限制提供了依据。     

Heterologous expression and purification of SpaB involved in subtilin biosynthesis

Lantibiotic peptides contain thioether bridges termed lanthionines that are putatively generated by dehydration of Ser and Thr residues followed by Michael addition of cysteine residues within the peptide. The LanB and LanC proteins have been proposed to catalyze the dehydration and formation of the thioether rings, respectively. We report here the first heterologous overexpression in Escherichia coli of SpaB, the putative dehydratase for subtilin. Sequence analysis of spaB revealed several nucleotide differences with current gene database entries. The solubility of SpaB was increased dramatically when co-expressed with GroEL/ES, and soluble His(6)-tagged SpaB was purified. The protein is at least a dimer, and interaction between SpaB and SpaC was observed. SpaS the putative substrate for SpaB was overexpressed in E. coli as an intein fusion protein, and after cleavage, the peptide was obtained in good yield.     

Synthesis by native chemical ligation and characterization of the scorpion toxin AmmTx3

The scorpion toxin AmmTx3 is a specific blocker of K_v4 channels. It was shown to have interesting potential for neurological disorders. In this study, we report the first chemical synthesis of AmmTx3 by using the native chemical ligation strategy and validate its biological activity. We determined its 3D structure by nuclear magnetic resonance spectroscopy, and pointed out that AmmTx3 possesses the well-known CSαβ structural motif, which is found in a large number of scorpion toxins. Overall, this study establishes an easy synthetic access to biologically active AmmTx3 toxin.     

Total chemical synthesis of dengue 2 virus capsid protein via native chemical ligation: Role of the conserved salt-bridge

The dengue capsid protein C is a highly basic alpha-helical protein of ～100 amino acid residues that forms an emphipathic homodimer to encapsidate the viral genome and to interact with viral membranes. The solution structure of dengue 2 capsid protein C (DEN2C) has been determined by NMR spectroscopy, revealing a large dimer interface formed almost exclusively by hydrophobic residues. The only acidic residue (Glu87) conserved in the capsid proteins of all four serotypes of dengue virus forms a salt bridge with the side chains of Lys45 and Arg55′. To understand the structural and functional significance of this conserved salt bridge, we chemically synthesized an N-terminally truncated form of DEN2C (^WTDEN2C) and its salt bridge-void analog ^E87ADEN2C using the native chemical ligation technique developed by Kent and colleagues. Comparative biochemical and biophysical studies of these two synthetic proteins using circular dichroism spectroscopy, fluorescence polarization, protein thermal denaturation, and proteolytic susceptibility assay demonstrated that the conserved salt bridge contributed to DEN2C dimerization and stability as well as its resistance to proteolytic degradation. Our work provided insight into the role of a fully conserved structural element of the dengue capsid protein C and paved the way for additional functional studies of this important viral protein.     

Metallic Zinc Reduction of Disulfide Bonds between Cysteine Residues in Peptides and Proteins

The use of powdered metallic zinc in acidic solution for the reduction of disulfide bonds in peptides and proteins has been investigated. The method has several advantages over the traditional mercapto based reducing methods currently used; the reducing agent is readily available and inexpensive; reduction can be performed in weakly acidic solutions of water and/or acetonitrile; work up simply consists of a centrifugation step followed by pipeting the supernatant from the metal pellet, thereby greatly diminishing the risk of reoxidation as a more elaborate work up procedure could result in. As no mercapto compounds are added, there is no risk that the reducing agent will interfere in subsequent modification of the thiol functionality. Disulfides in a model peptide are reduced within 5 min in any mixture of water/acetonitrile containing 1% TFA, all disulfides in insulin is reduced within 1 h in any mixture of water/acetonitrile containing 5% acetic acid. To stress the convenience of the metallic zinc reduction method, the resulting thiol compound was subjected to two commonly employed reactions in peptide chemistry: Cys(Npys) directed disulfide formation (70% yield) and native chemical ligation between the reduced model peptide and Boc-Ala-p-metylthiobenzyl ester (65% yield of the ligation product plus disulfide formation between Cys and p-thiocresol).     

Scope and limitation of side‐chain assisted ligation

Side‐chain assisted ligation is an auxiliary‐mediated ligation strategy in which a thiol bearing cyclohexane or cyclopentane is attached to the side‐chain of Asp, Glu, Ser or Thr to function in a similar manner to Cys in a native chemical ligation. Following the ligation step, the auxiliary is removed, without product isolation, under alkaline conditions. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

蛋白的色谱复性及同时纯化

对近年来新发展的用液相色谱(LC)进行蛋白质复性及同时纯化的方法做了评述,详细介绍了蛋白质在4种液相色谱上的复性及同时纯化的方法、设备和影响因素,并对各自的优缺点进行了比较,为色谱法作为研究蛋白质折叠及用于基因工程生产治疗蛋白质的复性及同时纯化技术的进一步应用提供依据。     

人巨细胞病毒pp150蛋白片段与MDBP蛋白片段的融合表达及初步应用

人巨细胞病毒(HCMV)感染是临床上常见的一种病毒性传播疾病,正常人群常无明显的临床症状,而对器官移植患者、免疫力低下及孕妇等人可产生严重的危害。以HCMVAD169病毒株基因为模板,经PCR扩增了编码pp150蛋白片段的UL32基因和编码MDBP蛋白片段的UL57基因,目的基因转化入pMD18-T克隆载体后再经酶切与表达载体pET-11a连接构建出融合基因表达载体,然后转入大肠杆菌BL21,重组大肠杆菌经诱导表达融合蛋白pp150/MDBP。经SDS-PAGE分析,其相对分子量约为27kD,表达量约占菌体蛋白的17.45%,Westernblot鉴定为阳性,ELISA及蛋白芯片检测表明融合蛋白具有良好的抗原性,经过初步应用表明其对血清IgG及IgM的检出率与全抗原相比一致,具有进一步开发应用的价值。     

FOXO 蛋白的修饰与细胞凋亡和癌变

Fox 蛋白家族是 2000 年才发布统一命名的蛋白质家族 . 由于其在生物体内所起的重要作用,已迅速成为生命科学研究的热点 . 目前,已经发现其家族成员有 100 多种,其中, FOXO 亚家族在动物细胞的凋亡中起重要作用 . 细胞凋亡与动物的长寿、繁殖、代谢、肿瘤发生及免疫有重要关系,对 Forkhead (Fox) 蛋白分子的命名进行了回顾、对其分类与结构特征进行了总结,并重点对 FOXO 的化学修饰、活性调节及其在细胞凋亡和肿瘤发生中的作用进行了综述 .     

Stability and fluctuations of amide hydrogen bonds in a bacterial cytochrome c: a molecular dynamics study

Molecular dynamics (MD) simulations on a bacterial cytochrome c were performed to investigate the lifetime and fluctuations of backbone hydrogen bonds and to correlate these data with protection factors for hydrogen exchange measured by NMR spectroscopy (Bartalesi et al. in Biochemistry, 42:10923–10930, 2003). The MD simulations provide a consistent pattern in that long lifetimes of hydrogen bonds go along with small amplitude fluctuations. In agreement with experiments, differences in stability were found with a rather flexible N-terminal segment as compared with a more rigid C-terminal part. Protection factors of backbone hydrogen exchange correlate strongly with the number of contacts but also with hydrogen-bond occupancy, hydrogen-bond survival times, as well as the inverse of fluctuations of backbone atoms and hydrogen-bond lengths derived from MD simulation data. We observed a conformational transition in the C-terminal loop, and significant motion in the N-terminal loop, which can be interpreted as being the structural units involved in the onset of the protein unfolding process in agreement with experimental evidence on mitochondrial cytochrome c. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Gernot Kieseritzky and Giulia Morra both contributed equally to this work.