InBase: the Intein Database

Inteins are self-catalytic protein splicing elements. InBase (http://www.neb.com/neb/inteins.html), the Intein Database and Registry, is a curated compilation of published and unpublished information about protein splicing. It presents general information as well as detailed data for each intein, including tabulated comparisons and a comprehensive bibliography. An intein-specific BLAST server is now available to assist in identifying new inteins.     

InBase, the New England Biolabs Intein Database.

Inteins are intervening sequences that splice as proteins, not RNA. InBase, the New England Biolabs Intein Database (http://www.neb. com/neb/inteins.html), is a comprehensive on-line database that includes the Intein Registry, along with detailed information about each intein and its host protein, tabulated comparisons and a comprehensive bibliography including papers in press.     

蛋白内含子与蛋白剪接

蛋白内含子和蛋白剪接是蛋白质研究的前沿领域。重点介绍了蛋白内含子的结构和蛋白剪接机理的最新研究成果 ;蛋白内含子如同RNA剪接中的内含子 ,也是一类可移动的遗传元件 ;蛋白内含子目前研究的热点是蛋白内含子的功能研究及其在蛋白质工程和其它生物工程领域的用。     

蛋白质内含子的研究进展

自1990年发现第一个蛋白质内含子以来,对其研究愈加引起注意。蛋白质内含子是蛋白质剪接元件,可从前体蛋白中切除并将两侧外显子连接起来成为成熟蛋白质,标准的蛋白质剪接主要包括四步亲核置换反应,新近又发现一种反式剪接机制。蛋白质内含子的演化形成存在先天遗传和后天插入两种方式,但目前还没有直接的实验证据。数据库显示,蛋白质内含子有10个保守模体：A、N2、B、N4、C、D、E、H、F和G,它们在蛋白质剪接过程中具有不同的作用。作为蛋白质剪切元件的蛋白质内含子,是蛋白质工程中一个功能强大的工具,具有重要的实践意义。现对蛋白质内含子的命名、分布、结构、剪接方式以及应用前景等作一全面的综述。     

The Mycobacterium avium-intracellulare complex dnaB locus and protein intein splicing

Intein is a protein sequence mebedded in-frame within a precursor protein and is posttranslationally excised by a self-catalytic protein splicing process. Protein splicing is believed to follow a pathway requiring Cys, Ser, or Thr residues at the intein N-terminus and substitutions other than Cys, Ser, or Thr residues prevent splicing. We show that the dnaB locus in some strains of M. avium-intracellulare complex (MAC) contains intein and that the intein N-terminal amino acid is Ala [Ala-type]. We demonstrate that the M. avium DnaB precursor protein undergoes posttranslational proteolytic processing producing proteins corresponding to the sizes of the DnaB and intein. Further, by Western analysis we detect a protein corresponding to the size of the spliced DnaB protein in MAC cell extracts. Together, these results indicate that the Ala-type MAC DnaB inteins can splice and provide another example that points to an interesting alternative splicing mechanism (Southworth, M. W., Benner, J., and Perler, F. B., EMBO J. 19, 5019-5026, 2000).     

内含肽介导的生物学效应及其应用

蛋白质翻译产物在成熟过程中剪切释放出来的一段氨基酸序列称为“intein”---即内含肽。它与前体蛋白以框内融合的形式共同翻译,并内嵌于前体蛋白序列中。内含肽的解离以及内含肽两侧氨基酸序列的连接是在内含肽自身催化作用下完成的。本文将从内含肽的发现、结构特征和作用机理等方面对这种具有特殊意义的蛋白质成熟机制进行较为全面的论述,同时介绍了近年来发展起来的以内含肽介导的蛋白质剪接为基础的蛋白质纯化和改造技术。     

Intein-containing chimeric protein construction and evaluation of its cleavage conditions

Protein splicing is a post-translational autocatalytic process that results in excision of internal peptide (intein) from a precursor protein and the ligation of the flanking protein sequences (exteins). High specificity of the intein-mediated excision of protein precursors allows the use of protein splicing in biotechnology. This work was aimed at the obtaining of human growth hormone with a native N-terminus in E. coli. Chimerical protein consisting of a short N-terminal peptide, Mxe GyrA intein and human growth hormone was created. During the translation formyl-methionine modified N-terminal peptide should have been removed by splicing. Intein was shown to mediate the cleavage of exteins, but their subsequent ligation was not observed. That allowed the preparation of human growth hormone with a native N-terminus. The effect of various factors on cleavage efficiency was studied. The most efficient cleavage of chimeric protein (60-80%) was achieved in the presence of inductor (100 mM beta-mercaptoethanol) upon the incubation for 4-6 days.     

Regulation of protein activity with small-molecule-controlled inteins

Inteins are the protein analogs of self-splicing RNA introns, as they post-translationally excise themselves from a variety of protein hosts. Intein insertion abolishes, in general, the activity of its host protein, which is subsequently restored upon intein excision. These protein elements therefore have the potential to be used as general molecular "switches" for the control of arbitrary target proteins. Based on rational design, an intein-based protein switch has been constructed whose splicing activity is conditionally triggered in vivo by the presence of thyroid hormone or synthetic analogs. This modified intein was used in Escherichia coli to demonstrate that a number of different proteins can be inactivated by intein insertion and then reactivated by the addition of thyroid hormone via ligand-induced splicing. This conditional activation was also found to occur in a dose-dependent manner. Rational protein engineering was then combined with genetic selection to evolve an additional intein whose activity is controlled by the presence of synthetic estrogen ligands. The ability to regulate protein function post-translationally through the use of ligand-controlled intein splicing will most likely find applications in metabolic engineering, drug discovery and delivery, biosensing, molecular computation, as well as many additional areas of biotechnology.     

Inteins, valuable genetic elements in molecular biology and biotechnology

Inteins are internal protein elements that self-excise from their host protein and catalyze ligation of the flanking sequences (exteins) with a peptide bond. They are found in organisms in all three domains of life, and in viral proteins. Intein excision is a posttranslational process that does not require auxiliary enzymes or cofactors. This self-excision process is called protein splicing, by analogy to the splicing of RNA introns from pre-mRNA. Protein splicing involves only four intramolecular reactions, and a small number of key catalytic residues in the intein and exteins. Protein-splicing can also occur in trans. In this case, the intein is separated into N- and C-terminal domains, which are synthesized as separate components, each joined to an extein. The intein domains reassemble and link the joined exteins into a single functional protein. Understanding the cis- and trans-protein splicing mechanisms led to the development of intein-mediated protein-engineering applications, such as protein purification, ligation, cyclization, and selenoprotein production. This review summarizes the catalytic activities and structures of inteins, and focuses on the advantages of some recent intein applications in molecular biology and biotechnology.     

一个新的蛋白质剪切系统及其剪切条件的研究

将含麦芽糖结合蛋白－内蛋白子－几丁质结合区（简称ＭＹＢ）基因的重组质粒ｐＭＹＢ１２９转入Ｅ．ｃｏｌｉ２４２６,在ＬＢ培养基中发酵,ＩＰＴＧ低温诱导表达,菌体超声破碎,离心,上清液经直链淀粉糖亲和层析,获得ＳＤＳ－ＰＡＧＥ电泳纯的前体蛋白ＭＹＢ．ＭＹＢ中内蛋白子的Ｎ端可被还原剂ＣｙＳＨ、ＤＴＴ、β－巯基乙醇等诱导发生剪切反应．结果表明,三种还原剂中以ＤＴＴ为最佳．同时对剪切过程中温度、ｐＨ值等影响进行了研究     

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.     

NMR structure of a KlbA intein precursor from Methanococcus jannaschii

Certain proteins of unicellular organisms are translated as precursor polypeptides containing inteins (intervening proteins), which are domains capable of performing protein splicing. These domains, in conjunction with a single residue following the intein, catalyze their own excision from the surrounding protein (extein) in a multistep reaction involving the cleavage of two intein-extein peptide bonds and the formation of a new peptide bond that ligates the two exteins to yield the mature protein. We report here the solution NMR structure of a 186-residue precursor of the KlbA intein from Methanococcus jannaschii, comprising the intein together with N- and C-extein segments of 7 and 11 residues, respectively. The intein is shown to adopt a single, well-defined globular domain, representing a HINT (Hedgehog/Intein)-type topology. Fourteen beta-strands are arranged in a complex fold that includes four beta-hairpins and an antiparallel beta-ribbon, and there is one alpha-helix, which is packed against the beta-ribbon, and one turn of 3(10)-helix in the loop between the beta-strands 8 and 9. The two extein segments show increased disorder, and form only minimal nonbonding contacts with the intein domain. Structure-based mutation experiments resulted in a proposal for functional roles of individual residues in the intein catalytic mechanism.     

Modular organization of inteins and C-terminal autocatalytic domains.

Analysis of the conserved sequence features of inteins (protein "introns") reveals that they are composed of three distinct modular domains. The N-terminal (N) and C-terminal (C) domains are predicted to perform different parts of the autocatalytic protein splicing reaction. An optional endonuclease domain (EN) is shown to correspond to different types of homing endonucleases in different inteins. The N domain contains motifs predicted to catalyze the first steps of protein splicing, leading to the cleavage of the intein N terminus from its protein host. Intein N domain motifs are also found in C-terminal autocatalytic domains (CADs) present in hedgehog and other protein families. Specific residues in the N domain of intein and CADs are proposed to form a charge relay system involved in cleaving their N-termini. The intein C domain is apparently unique to inteins and contains motifs that catalyze the final protein splicing steps: ligation of the intein flanks and cleavage of its C terminus to release the free intein and spliced host protein. All intein EN domains known thus far have dodecapeptide (DOD, LAGLI-DADG) type homing endonuclease motifs. This work identifies an EN domain with an HNH homing-endonuclease motif and two new small inteins with no EN domains. One of these small inteins might be inactive or a "pseudo intein." The results suggest a modular architecture for inteins, clarify their origin and relationship to other protein families, and extend recent experimental findings on the functional roles of intein N, C, and EN motifs.     

REBASE-restriction enzymes and methylases.

REBASE is a comprehensive database of information about restriction enzymes and their associated methylases, including their recognition and cleavage sites and their commercial availability. Information from REBASE is available via monthly electronic mailings as well as via anonymous ftp, WAIS/gopher and through the World Wide Web (http://www.neb.com/rebase ). Specialized files are available that can be used directly by many software packages.     

REBASE: restriction enzymes and methyltransferases

REBASE contains comprehensive information about restriction enzymes, DNA methyltransferases and related proteins such as nicking enzymes, specificity subunits and control proteins. It contains published and unpublished references, recognition and cleavage sites, isoschizomers, commercial availability, crystal and sequence data. Homing endonucleases are also included. REBASE contains the most complete and up-to-date information about the methylation sensitivity of restriction endonucleases. In addition, there is extensive information about the known and putative restriction-modification (R-M) systems in more than 100 sequenced bacterial and archaeal genomes. The data is available on the web (http://rebase.neb.com/rebase/rebase.html), through ftp (ftp.neb.com) and as monthly updates via email.     

REBASE--restriction enzymes and methylases.

REBASE is a comprehensive database of information about restriction enzymes and their associated methylases, including their recognition and cleavage sites and their commercial availability. Information from REBASE is available via monthly electronic mailings as well as via WAIS, anonymous ftp and through the World Wide Web (http://www.neb.com/rebase). Specialized files are available that can be used directly by many software packages.     

NEBcutter: A program to cleave DNA with restriction enzymes

NEBcutter, version 1.0, is a program available via a web server (http://tools.neb.com/NEBcutter) that will accept an input DNA sequence and produce a comprehensive report of the restriction enzymes that will cleave the sequence. It produces a variety of outputs including restriction enzyme maps, theoretical digests and links into the restriction enzyme database, REBASE (http://www.neb.com/rebase). Importantly, its table of recognition sites is updated daily from REBASE and it marks all sites that are potentially affected by DNA methylation (Dam, Dcm, etc.). Many options exist to choose the enzymes used for digestion, including all known specificities, subsets of those that are commercially available or sets of enzymes that produce compatible termini.     

Chimeric spider silk proteins mediated by intein result in artificial hybrid silks

Hybrid silks hold a great potential as specific biomaterials due to its controlled mechanical properties. To produce fibers with tunable properties, here we firstly made chimeric proteins in vitro, called W2C4CT and W2C8CT, with ligation of MaSp repetitive modules (C) with AcSp modules (W) by intein trans splicing technology from smaller precursors without final yield reduction. Intein mediated chimeric proteins form fibers at a low concentration of 0.4 mg/mL in 50 mM K₃PO₄ pH 7.5 just drawn by hand. Hybrid fibers show smoother surface, and also have stronger chemical resistance as compared with fibers from W2CT (W fibers) and mixture of W2CT/C8CT (MHF8 fibers). Fibers from chimeric protein W2C4CT (HFH4) have improved mechanical properties than W fibers; however, with more C modules W2C8CT fibers (HFH8) properties decreased, indicates the length proportion of various modules is very important and should be optimized for fibers with specific properties. Generally, hybrid silks generated via chimeric proteins, which can be simplified by intein trans splicing, has greater potential to produce fibers with tunable properties. Our research shows that intein mediated directional protein ligation is a novel way to make large chimeric spider silk proteins and hybrid silks. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 385–392, 2016.     

REBASE - restriction enzymes and methylases

REBASE is a comprehensive database of information about restriction enzymes and related proteins. It contains published and unpublished references, recognition and cleavage sites, isoschizomers, commercial availability, methylation sensitivity, crystal and sequence data. DNA methyltransferases, homing endonucleases, nicking enzymes, specificity subunits and control proteins are also included. Most recently, putative DNA methyltransferases and restriction enzymes, as predicted from analysis of genomic sequences, are also listed. The data is distributed via Email, ftp (ftp.neb.com), and the Web (http://rebase.neb.com).     

REBASE--restriction enzymes and methylases

REBASE contains comprehensive information about restriction enzymes, DNA methylases and related proteins such as nicking enzymes, specificity subunits and control proteins. It contains published and unpublished references, recognition and cleavage sites, isoschizomers, commercial availability, methylation sensitivity, crystal data and sequence data. Homing endonucleases are also included. Most recently, extensive information about the methylation sensitivity of restriction enzymes has been added and a new feature contains complete analyses of the putative restriction systems in the sequenced bacterial and archaeal genomes. The data is distributed via email, ftp (ftp.neb.com) and the Web (http://rebase. neb.com).