高剪接活性断裂蛋白质内含子的体内切割

蛋白质内含子介导的断裂(切割)反应被用于蛋白质纯化、连接和环化等,但目前仍存在断裂效率低、断裂反应的不可控、产物复杂等问题。蛋白质内含子的定点突变可导致其N端或C端断裂。其末位氨基酸突变则剪接反应第3步天冬酰胺环化无法进行,发生N端断裂;其首位氨基酸发生突变则剪接反应第一步酰基重排及其后续步骤均无法进行,而天冬酰胺环化仍可进行,发生C端断裂。利用已获得的高剪接活性的S1和S11型断裂蛋白质内含子Ssp GyrB,分别将其参与剪接反应的首位半胱氨酸或末位天冬酰胺突变为丙氨酸,构建能够发生一端断裂的断裂蛋白质内含子。研究结果表明,突变后断裂蛋白质内含子的剪接反应几乎不发生,其断裂活性有不同程度的提高,获得了在大肠杆菌体内具有较高效断裂活性的断裂蛋白质内含子。这将为进一步研究其体外可控性剪接、构建高效的蛋白纯化系统和深入研究蛋白质内含子的剪接机制提供基础。     

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.     

新型断裂内含肽介导的可控性C端断裂

内含肽介导的蛋白质断裂被广泛地应用于蛋白质纯化、连接和环化. 但目前的方法都是用传统的连续的内含肽来介导蛋白质断裂反应,因而往往存在自发性断裂、产率低等问题. 本实验选择3个S1型新型断裂内含肽Ter ThyX、Ssp GryB和Rma DnaB来实现蛋白质断裂反应的可控性. 在可控性C端断裂反应中,S1型断裂内含肽的C端片段（IC ）与硫氧还蛋白（T）融合作为前体蛋白,加入化学合成的Ssp DnaB S1型断裂内含肽 的N端小肽与二硫苏糖醇（DTT）共同诱导C端断裂反应.结果表明,该小肽可以诱导这 3个不同的S1型断裂内含肽的前体蛋白发生C端断裂反应. 该方法为利用内含肽C端断 裂介导的蛋白质纯化提供了更多的选择,并为内含肽的结构与功能的关系研究提供-有用的线索.     

蛋白质内含子在特定氨基酸序列中的剪接

基因工程技术已经被广泛应用于抗体的生产。但是由于抗体的分子量较大,导致合成抗体较为困难。蛋白质内含子是前体蛋白质中的一段氨基酸序列,能够将自身剪切出来,并将两端的外显子连接形成成熟的蛋白质。将抗体的Fab(antigen binding fragment)和Fc(crystalline fragment)分别与蛋白质内含子(intein) 的N端(IN)和C端(IC)融合表达,利用蛋白质内含子的剪接功能,可形成完整的抗体分子。KSCDKTH是存在于抗体铰链区(hinge region)的一段氨基酸序列,如果在KSCDKTH序列中筛选到高效剪接的蛋白质内含子,即可通过蛋白质剪接,将抗体分子的Fab和Fc剪接形成完整抗体。本文筛选发现,Ssp DnaX的3种断裂蛋白质内含子(S0, S1, S11)具有在KSCDKTH序列中高效剪接的能力,这一研究结果为抗体的剪接合成提供了可行性。     

断裂蛋白质内含子的剪接机制、起源和进化

蛋白质内含子(intein)是具有自我催化活性的蛋白质. 翻译后,通过蛋白质剪接从蛋白质前体中去掉,并以肽键连接两侧蛋白质外显子(extein)形成成熟蛋白质. 断裂蛋白质内含子(split intein)在蛋白质内含子中部区域特定位点发生断裂,形成N端片段和C端片段,分别由基因组上相距较远的两个基因编码. 现在已知,它仅分布于蓝细菌和古细菌中. 断裂蛋白质内含子的N端片段和C端片段通过非共价键(如静电作用)相互识别,重建催化活性中心,介导蛋白质反式剪接. 断裂蛋白质内含子的发现进一步深化了人们对基因表达和蛋白质翻译后成熟过程复杂性的认识,而且它在蛋白质工程、蛋白质药物开发和蛋白质结构与功能研究等方面有非常广泛的应用. 本文试图综述断裂蛋白质内含子的分布、结构特征和剪接机制,并分析其可能的起源和进化途径.     

血管生成素与tRNA片段化

tRNA主要功能是转运氨基酸参与蛋白质合成,在蛋白质生物合成过程中起着关键性的作用．近年来发现,tRNA是细胞内小RNA分子的重要来源,具有其它重要的生物学功能．来源于成熟tRNA分子的tRNA片段根据切割位置及生成机制的不同,主要分为两类：一类是tRNA半分子（tRNA halves）;另一类是较小的tRNA片段,称为tRFs( tRNA fragments)．在哺乳动物细胞中,tRNA半分子由血管生成素在tRNA分子反密码环处切割生成．本文主要针对tRNA半分子的加工机制、功能及在临床上的潜在应用进行综述．     

多种S1断裂内含肽的体内交叉反应

断裂内含肽含有两个独立分离的多肽片段(N端内含肽和C端内含肽),它催化蛋白质反式剪接反应,在蛋白质研究与蛋白质工程中已得到诸多实际应用.在蛋白质反式剪接过程中,内含肽的N端内含肽和C端内含肽通过结构互补特异性地非共价组合.然而,Ssp DnaX S1型断裂内含肽的较大C端内含肽片段近来被发现能够与源自其它内含肽的N端内含肽片段交叉反应,表明蛋白质内含子Ssp DnaX具有结构杂交特征.本研究对另外2种S1型内含肽Rma DnaB和Ssp GyrB的较大C端内含肽与不同S1型断裂内含肽的N 端内含肽交叉反应活性进行分析检测.目的是探讨S1型断裂内含肽的结构杂交特征是否具有普遍性.结果发现,Rma DnaB的S1 C端内含肽能够与Ssp GyrB的S1 N端内含肽交叉反应,却不能与Ssp DnaX的S1 N端内含肽交叉反应;与此相似,Ssp GyrB的S1 C端内含肽能够与Rma DnaB的 S1 N端内含肽交叉反应,却不能与Ssp DnaX的S1 N端内含肽交叉反应.此外,某些交叉反应表现出温度依赖性.这些结果对于内含肽的结构 功能关系以及S1型断裂内含肽的应用研究具有重要的意义.     

应用N端Cys-free断裂蛋白质内含子标记蛋白质N端

蛋白质的特异位点修饰可以帮助了解蛋白质的结构与功能.但是,现有的蛋白质特异位点标记方法种类有限,而且存在局限性,所以有必要开发新的蛋白质特异位点标记方法．以谷胱甘肽-S-转移酶(GST)为研究对象,借助蛋白质反式剪接技术,建立了利用新型断裂蛋白质内含子对蛋白质进行N 端标记的新方法.在这个方法中,通过简单的重组表达、标记和纯化得到带有荧光基团的小肽,经过蛋白质反式剪接,荧光基团被标记到蛋白质的N 端. 初步研究结果显示,标记效率可达到12 %.     

The cleavage program in the 2d cell lineage of Tubifex embryos

Early development in clitellate annelids is characterized by a highly stereotyped sequence of unequal, spiral cleavages. Cell 2d (i.e., the second micromere of the D quadrant) in the oligochaete Tubifex tubifex also undergoes an evolutionarily conserved sequence of cell division to produce four bilateral pairs of ectodermal teloblasts that act as embryonic stem cells. This study was conducted to characterize each of the 15 rounds of cell division that occur in the 2d cell lineage in this clitellate. After its occurrence, cell 2d undergoes three rounds of highly unequal divisions, giving off the first smaller daughter cell toward the posterior right of the larger daughter cell, the second cell toward the posterior left, and the third cell toward the anterior side of the cell; the larger daughter cell that results from the third division (i.e., the great-granddaughter cell of 2d) then divides equally into a bilateral pair of NOPQ proteloblasts. Cell NOPQ on either side of the embryo undergoes 11 rounds of cell division, during which ectoteloblasts N, Q, and O/P are produced in this order. After its appearance, NOPQ undergoes highly unequal divisions twice cutting off the smaller cells toward the anterior end of the embryo and then divides almost equally into ectoteloblast N and proteloblast OPQ. After its appearance, OPQ undergoes highly unequal divisions twice giving off the first smaller cell toward the anterior and the second smaller cell toward the posterior of the embryo and then divides almost equally into ectoteloblast Q and proteloblast OP. Finally, OP undergoes highly unequal division four times after its birth budding off the smaller cells toward the anterior and then cleaves equally into ectoteloblasts O and P. In the unequally dividing cells of the 2d cell lineage, the mitotic apparatus (MA), which forms at the cell's center, moves eccentrically toward the cortical site where the smaller cell will be given off. The moving MA is oriented perpendicular to the surface it approaches, and its peripheral pole becomes closely associated with the cell cortex. In contrast, the MA involved in the equal divisions remains in the cell center throughout mitosis. The key features of the cleavage program in the 2d cell lineage are discussed in light of the present observations. The mechanical aspects of unequal cleavage in the 2d cell lineage and the modes of specification of MA orientation are discussed. A comparison of the cleavage mode in the 2d cell lineage is also performed among six selected clitellate annelid species.     

An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1–3)insulin-like growth factor I

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1–3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1–3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1–3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.     

Synthesis, characterization, and C-H/C-C cleavage reactions of two rhodium-trispyrazolylborate dihydrides

Treatment of Tp′Rh(PMe₃)Cl₂ and Tp′Rh(CNCH₂CMe₃)Cl₂ with Cp₂ZrH₂ produces Tp′Rh(PMe₃)H₂ and Tp′Rh(CNCH₂CMe₃)H₂, respectively, in excellent yield. Photolysis of benzene solutions of each dihydride complex generates hydrogen and the fragment [Tp′Rh(L)] which inserts into the solvent C-H bond. The phosphine dihydride has also been shown to be a catalyst for the hydrogenation of biphenylene, showing a capability to cleave C-C bonds. Reductive elimination of benzene from Tp′Rh(PMe₃)PhH is nearly 250 times slower than from Cp*Rh(PMe₃)PhH.     

A novel 1,2,4-triazole-based copper(II) complex: Synthesis, characterization, magnetic property and nuclease activity

A novel binuclear copper(II) complex [Cu₂L(μ-SO₄)](PF₆)₂ (1) (L = 3,5-bis (bis(pyridine-2-ylmethyl)amino)methyl)-4H-1,2,4-triazol-4-amine) has been synthesized and structurally characterized. X-ray structure shows that the two copper(II) atoms are bridged by one bidentate sulfate ion and the 1,2,4-triazole ring of L with Cu1?Cu2 distance of 4.404 Å. Each copper(II) center has a distorted trigonal-bipyramidal configuration. Variable-temperature magnetic susceptibility studies (2-300 K) indicate the existence of weak antiferromagnetic coupling between the copper(II) ions in complex 1. The interaction of complex 1 with calf thymus DNA (CT-DNA) has been studied by UV absorption, fluorescence spectroscopy, circular dichroism spectroscopy, viscosity and cyclic voltammetry. Furthermore, complex 1 was able to promote single and double strand DNA cleavage in both aerobic and anaerobic conditions, the pseudo-Michaelis-Menten kinetic parameters k_cat = 2.58 h⁻¹ and K_m = 1.2 × 10⁻⁴ M were obtained for 1. The hydrolytic cleavage of DNA by the complex was supported by the evidence from free radical quenching, anaerobic experiment, thiobarbituric acid-reactive substances (TBARS) assay.     

Kinetics of circular DNA molecule digestion by restriction endonuclease Computation of kinetic constants from time dependence of fragment concentrations

A model for kinetics of circular substrate cleavage by restriction endonuclease was formulated. The aim of the analysis of the model was to extract kinetic constants for all target sites from time-dependence of fragment concentration in reaction products. That was proved to be possible for molecules with an odd number of fragments only. A symmetry of the molecules with an even number of fragment is the cause. A solution for molecules with an odd number of fragments was found and methods for dealing with the other molecules were suggested.Preliminary results were presented at VIIth CMEA Symposium Biophysics of Nucleic Acids and Proteins, Brno (Czechoslovakia) December 2–6, 1985.     

The unique developmental program of the acoel flatworm, Neochildia fusca

Acoel embryos exhibit a unique form of development that some investigators argue is related to that found in polyclad turbellarians and coelomate spiralians, which display typical quartet spiral cleavage. We generated the first cell-lineage fate map for an acoel flatworm, Neochildia fusca, using modern intracellular lineage tracers to assess the degree of similarity between these distinct developmental programs. N. fusca develops via a "duet" cleavage pattern in which second cleavage occurs in a leiotropically oblique plane relative to the animal-vegetal axis. At the four-cell stage, the plane of first cleavage corresponds to the plane of bilateral symmetry. All remaining cleavages are symmetrical across the sagittal plane. No ectomesoderm is formed; the first three micromere duets generate only ectodermal derivatives. Endomesoderm, including the complex assemblage of circular, longitudinal, and oblique muscle fibers, as well as the peripheral and central parenchyma, is generated by both third duet macromeres. The cleavage pattern, fate map, and origins of mesoderm in N. fusca share little similarity to that exhibited by other spiralians, including the Platyhelminthes (e.g., polyclad turbellarians). These findings are considered in light of the possible evolutionary origins of the acoel duet cleavage program versus the more typical quartet spiral cleavage program. Finally, an understanding of the cell-lineage fate map allows us to interpret the results of earlier cell deletion studies examining the specification of cell fates within these embryos and reveals the existence of cell-cell inductive interactions in these embryos.     

A variant in the restriction endonuclease cleavage pattern of mitochondrial DNA in the domestic fowl, Gallus gallus domesticus

Summary. The cleavage patterns of mitochondrial DNAs (mtDNAs) were investigated from 15 lines of domestic fowls, Gallus gallus domesticus . using 11 restriction endonucleases. The cleavage patterns with 10 restriction endonucleases were identical in all the lines. A variant was found in a line of White Leghorn in the pattern with Mspl digestions. Cleavage patterns of the red jungle fowl, Gallus gallus gallus , were identical to the common patterns shown by the 14 lines of domestic fowls.     

Generating recombinant C-terminal prion protein fragments of exact native sequence

Transmissibility and distinctive neuropathology are hallmark features of prion diseases differentiating them from other neurodegenerative disorders, with pathogenesis and transmission appearing closely linked to misfolded conformers (PrP(Sc)) of the ubiquitously expressed cellular form of the prion protein (PrP(C)). Given the apparent pathogenic primacy of misfolded PrP, the utilisation of peptides based on the prion protein has formed an integral approach for providing insights into misfolding pathways and pathogenic mechanisms. In parallel with studies employing prion peptides, similar approaches in other neurodegenerative disorders such as Alzheimer Disease, have demonstrated that differential processing of parent proteins and quite minor variations in the primary sequence of cognate peptides generated from the same constitutive processing (such as Aβ1-40 versus Aβ1-42 produced from γ-secretase activity) can be associated with very different pathogenic consequences. PrP(C) also undergoes constitutive α- or β-cleavage yielding C1 (residues 112-231 human sequence) or C2 (residues 90-231), respectively, with the full cell biological significance of such processing unresolved; however, it is noteworthy that in prion diseases, such as Creutzfeldt-Jakob disease (CJD) and murine models, the moderately extended C2 fragment predominates in the brain suggesting that the two cleavage events and the consequent C-terminal fragments may differ in their pathogenic significance. Accordingly, studies characterising biologically relevant peptides like C1 and C2, would be most valid if undertaken using peptides completely free of any inherent non-native sequence that arises as a by-product of commonly employed recombinant production techniques. To achieve this aim and thereby facilitate more representative biophysical and neurotoxicity studies, we adapted the combination of high fidelity Taq TA cloning with a SUMO-Hexa-His tag-type approach, incorporating the SUMO protease step. This technique consistently produced sufficient yields (～10 mg/L) of high purity peptides (>95%) equating to C1 and C2 of exact native primary sequence in the α-helical conformation suitable for biological and biophysical investigations.     

Plasmid-mediated degradation of o- and p-phthalate by Pseudomonas fluorescens

A Pseudomonas fluorescens strain SKP3 capable of utilizing both phthalic acid and terephthalic acid as sole source of carbon and energy was isolated by enrichment technique. Phthalic acid, terephthalic acid and protocatechuic acid were easily oxidized by both phthalate-grown and glucose-grown cells without a lag period. Phthalic acid is metabolized through the ortho cleavage pathway and terephthalic acid through the meta cleavage pathway and the enzymes of the two pathways are constitutive in nature. A large plasmid of approximately 140kb in size was found to be involved in the degradation of phthalates. The catabolic plasmid pSKL was transferable to different hosts.     

The relation of time to direction and equality of cleavage in Ilyanassa embryos

Summary

Cleavage inhibition experiments using cytochalasin B and hydrostatic pressure demonstrate the existence of a “clock mechanism” specifying cleavage time and form in Ilyanassa obsoleta embryos. Cytokinesis but not karyokinesis is inhibited during these treatments. When second cleavage is inhibited, the following cleavage occurs approximately on schedule with controls. Two micromeres are produced in this cleavage even though treated embryos consist of only two cells. When third cleavage is inhibited, the following micromere cleavage occurs in a counter clockwise direction, typical for the controls. Treatment with nocodazole, an antitubulin drug, inhibits both cytokinesis and karyokinesis but does not affect the cleavage clock mechanism. Treatment with 2,4-dinitrophenol stops both cleavage and the clock mechanism. These results indicate that the cleavage clock in Ilyanassa requires energy but does not depend on centrosomal behavior or on the form of previous cleavages. With regard to the production of micromeres the clock may involve an interaction between the aster-spindle complex and special regions of the animal pole cortex.