Semisynthesis of cytotoxic proteins using a modified protein splicing element.

Two cytotoxic proteins, bovine pancreatic ribonuclease A (RNase A), and a restriction endonuclease from Haemophilus parainfluenzae (HpaI), were produced using a novel semisynthetic approach that utilizes a protein splicing element, an intein, to generate a reactive thioester at the C-terminus of a recombinant protein. Nucleophilic attack on this thioester by the N-terminal cysteine of a synthetic peptide ultimately leads to the ligation of the two reactants through a native peptide bond. This strategy was used to produce RNase A and HpaI by isolating inactive truncated forms of these proteins, the first 109 and 223 amino acids of RNase A and HpaI, respectively, as fusion proteins consisting of the target protein, an intein, and a chitin binding domain. Thiol-induced cleavage of the precursor led to the liberation of the target protein with a C-terminal thioester-tag. Addition of synthetic peptides representing the amino acids missing from the truncated forms led to the generation of full-length products that displayed catalytic activity indicative of the wild-type enzymes. The turnover numbers and Km for ligated and renatured RNase A were 8.2 s(-1) and 1.5 mM, in good agreement with reported values of 8.3 s(-1) and 1.2 mM (Hodges & Merrifield, 1975). Ligated HpaI had a specific activity of 0.5-1.5 x 10(6) U/mg, which compared favorably with the expected value of 1-2 x 10(6) U/mg (J. Benner, unpubl. obs.). Besides assisting in the production of cytotoxic proteins, this technique could allow the easy insertion of unnatural amino acids into a protein sequence.     

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.     

Protein splicing

Protein splicing is a posttranslational process that results in excision of an internal protein region (intein) and ligation of its flanking sequences (exteins). As distinguished from other variants of protein processing, protein splicing does not require cofactors of enzymes. Protein splicing is catalyzed by an internal domain (so-called Hint domain) of the intein itself. The review considers the main regularities and molecular mechanisms of the process, as well as the functions of Hint domains in other protein families (Hh proteins, bacterial BIL domains, etc.). Studies of protein splicing are of importance from both theoretical and applied viewpoints. For instance, comparisons of the inteins found in different domains of life illustrate the role of horizontal transfer in intein spreading. A possible role of inteins in regulating several cell processes is discussed on the basis of recent data.     

The curious case of protein splicing: mechanistic insights suggested by protein semisynthesis.

The gradual accumulation of examples of protein splicing, in which a nested intervening sequence is spliced out of the interior of a polyprotein precursor, suggests that this curious phenomenon might prove to have universal phylogenetic distribution and biological significance. The known examples are reviewed, with the aim of establishing underlying patterns, and a generalized mechanism of autocatalytic protein splicing is proposed. The testable consequences of such a proposal and the possible evolutionary origins of the phenomenon are discussed.     

Improved segmental isotope labeling of proteins and application to a larger protein

A new isotope labeling technique for peptide segments in a protein sample was recently established using the protein splicing element intein [Yamazaki et al. (1998) J. Am. Chem. Soc., 120, 5591–5592]. This method makes it possible to observe signals of a selected amino (N-) or carboxyl (C-) terminal region along a peptide chain. However, there is a problem with the yield of the segmentally labeled protein. In this paper, we report an increase in the yield of the protein that enables the production of sufficient amounts of segmentally ¹³C/¹⁵N-labeled protein samples. This was achieved by improvement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation. The N-terminal fragment was expressed as a fused protein with the cellulose binding domain at its N-terminus, which was expressed as an insoluble peptide in cells and the expression level was increased. Incubation with 2.5 M urea and 50% glycerol increased the efficiency of the refolding greatly, thereby raising the final yields of the ligated proteins. The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the results for a maltose binding protein consisting of 370 amino acids. All four examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples.     

Engineered Ssp DnaX inteins for protein splicing with flanking proline residues

Inteins are internal protein sequences capable of catalyzing a protein splicing reaction by self-excising from a precursor protein and simultaneously joining the flanking sequences with a peptide bond. Split inteins have separate pieces (N-intein and C-intein) that reassemble non-covalently to catalyze a protein trans-splicing reaction joining two polypeptides. Protein splicing has become increasingly useful tools in many fields of biological research and biotechnology. However, natural and engineered inteins have failed previously to function when being flanked by proline residue at the −1 or +2 positions, which limits general uses of inteins. In this study, different engineered inteins were tested. We found that engineered Ssp DnaX mini-intein and split inteins could carry out protein splicing with proline at the +2 positions or at both −1 and +2 positions. Under in vivo conditions in E. coli cells, the mini-intein, S1 split intein, and S11 split intein spliced efficiently, whereas the S0 split intein did not splice with proline at both −1 and +2 positions. The S1 and S11 split inteins also trans-spliced efficiently in vitro with proline at the +2 positions or at both −1 and +2 positions, but the S0 split intein trans-spliced inefficiently with proline at the +2 position and did not trans-splice with proline at both −1 and +2 positions. These findings contribute significantly to the toolbox of intein-based technologies by allowing the use of inteins in proteins having proline at the splicing point.     

Crystal structures of an intein from the split dnaE gene of Synechocystis sp. PCC6803 reveal the catalytic model without the penultimate histidine and the mechanism of zinc ion inhibition of protein splicing

The first naturally occurring split intein was found in the dnaE gene of Synechocystis sp. PCC6803 and belongs to a subclass of inteins without a penultimate histidine residue. We describe two high-resolution crystal structures, one derived from an excised Ssp DnaE intein and the second from a splicing-deficient precursor protein. The X-ray structures indicate that His147 in the conserved block F activates the side-chain N(delta) atom of the intein C-terminal Asn159, leading to a nucleophilic attack on the peptide bond carbonyl carbon atom at the C-terminal splice site. In this process, Arg73 appears to stabilize the transition state by interacting with the carbonyl oxygen atom of the scissile bond. Arg73 also seems to substitute for the conserved penultimate histidine residue in the formation of an oxyanion hole, as previously identified in other inteins. The finding that the precursor structure contains a zinc ion chelating the highly conserved Cys160 and Asp140 reveals the structural basis of Zn2+-mediated inhibition of protein splicing. Furthermore, it is of interest to observe that the carbonyl carbon atom of Asn159 and N(eta) of Arg73 are 2.6 angstroms apart in the free intein structure and 10.6 angstroms apart in the precursor structure. The orientation change of the aromatic ring of Tyr-1 following the initial acyl shift may be a key switching event contributing to the alignment of Arg73 and the C-terminal scissile bond, and may explain the sequential reaction property of the Ssp DnaE intein.     

Trans protein splicing of cyanobacterial split inteins in endogenous and exogenous combinations

Inteins are autocatalytic protein domains that post-translationally excise from protein precursors and ligate their flanking regions with a peptide bond, in a process called protein splicing. Intein-containing DNA polymerases of cyanobacteria and nanoarchaea are naturally split into two separate genes at their intein domain. Such naturally occurring split inteins rapidly self-associate and reconstitute protein-splicing activity in trans. Here, we analyze the in vitro protein-splicing activity of three naturally split inteins from diverse cyanobacteria: Oscillatoria limnetica, Thermosynechococcus vulcanus, and Nostoc sp. PCC7120. N- and C-terminal halves of these split inteins were mixed in nine combinations, resulting in three endogenous (wild-type) and six exogenous combinations. Protein splicing was detected in all split-intein combinations, despite a 30-50% sequence variation between the homologous proteins. Splicing activity proceeded under a variety of conditions, including the presence of denaturants and reductants and high temperature, ionic strength, and viscosity. Still, in a high concentration of salt (2 M) or urea (6 M), specific combinations spliced significantly better than others. Additionally, copper ions were found to inhibit trans splicing in a reversible double-lock reaction. Our comparative analysis of naturally split inteins in endogenous and exogenous combinations demonstrates the modularity of trans protein-splicing elements and their robust activity. It suggests tight interactions between split-intein halves and conditions for modifying the specificity of intein parts. These results promote the biotechnological use of split inteins for controlled assembly of protein fragments either in vivo or in vitro and under moderate or extreme conditions.     

Protein splicing — the lengths some proteins will go to

We review the recently discovered phenomenon of protein splicing which is the excision of an internal protein sequence at the protein level rather than at the RNA level. The means by which examples of protein splicing have been identified are described, and the similarities of the internally spliced protein products (or inteins) are discussed. Comparisons are made between inteins and group I RNA introns. We describe the evidence supporting excision of intiens by a post-translational autocatalytic reaction of a full length polypeptide precursor, rather than by RNA splicing. An examination is made of some of the proposed mechanism schemes and the supporting them presented.     

Controllable protein cleavages through intein fragment complementation

Intein‐based protein cleavages, if carried out in a controllable way, can be useful tools of recombinant protein purification, ligation, and cyclization. However, existing methods using contiguous inteins were often complicated by spontaneous cleavages, which could severely reduce the yield of the desired protein product. Here we demonstrate a new method of controllable cleavages without any spontaneous cleavage, using an artificial S1 split‐intein consisting of an 11‐aa N‐intein (I_N) and a 144‐aa C‐intein (I_C). In a C‐cleavage design, the I_C sequence was embedded in a recombinant precursor protein, and the small I_N was used as a synthetic peptide to trigger a cleavage at the C‐terminus of I_C. In an N‐cleavage design, the short I_N sequence was embedded in a recombinant precursor protein, and the separately produced I_C protein was used to catalyze a cleavage at the N‐terminus of I_N. These N‐ and C‐cleavages showed >95% efficiency, and both successfully avoided any spontaneous cleavage during expression and purification of the precursor proteins. The N‐cleavage design also revealed an unexpected and interesting structural flexibility of the I_C protein. These findings significantly expand the effectiveness of intein‐based protein cleavages, and they also reveal important insights of intein structural flexibility and fragment complementation.     

SR蛋白家族在RNA剪接中的调控作用

SR蛋白家族成员都具有一个富含丝氨酸/精氨酸(S/R)重复序列的RS结构域,在RNA剪接体的组装和选择性剪接的调控过程中具有重要的作用。绝大多数SR蛋白是生存的必需因子,通过其RS结构域和特有的其他结构域,实现与前体mRNA的特异性序列或其他剪接因子的相互作用,协同完成剪接位点的正确选择或促进剪接体的形成。深入研究SR蛋白家族在RNA选择性剪接中的调控机制,可以促进以疾病治疗或害虫防治为目的的应用研究。该文总结了SR蛋白家族在基础研究和应用方面的进展。     

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.     

Split Gp41-1 intein splicing as a model to evaluate the cellular location of the oncosuppressor Maspin in an in vitro model of osteosarcoma

Inteins are proteins involved in the protein splicing mechanism, an autoprocessing event, where sequences (exteins) separated by inteins become ligated each other after recombination. Two kinds of inteins have been described, contiguous inteins and split inteins. The former ones are transcribed and translated as a single peptide along with their exteins, while the latter are fragmented between two different genes and are transcribed and translated separately. The aim of this study is to establish a method to obtain a fluorescent eukaryotic protein to analyze its cellular localization, using the natural split gp41-1 inteins. We chose natural split inteins due to their distribution in all three domains of life. Two constructs were prepared, one containing the N-terminal split intein along with the N-moiety of the Red Fluorescent Protein (RFP) and a second construct containing the C-terminal of split intein, the C-moiety of RFP and the gene coding for Maspin, a tumor suppressor protein. The trans-splicing was verified by transfecting both N-terminal and C-terminal constructs into mammalian cells. The success of the recombination event was highlighted through the fluorescence produced by reconstituted RFP after recombination, along with the overlap of the red fluorescence produced by recombined RFP and the green fluorescence produced by the hybridization of the recombinant Maspin with a specific antibody. In conclusion, we opted to use this mechanism of recombination to obtain a fluorescent Maspin instead to express a large fusion protein, considering that it could interfere with Maspin's structure and function.     

Protein splicing of the three Pyrococcus abyssi ribonucleotide reductase inteins

An intein is a polypeptide that interrupts the functional domains of a protein, called the exteins. The intein can facilitate its own excision from the exteins, concomitant with the ligation of the exteins, in a process called protein splicing. The alpha subunit of the ribonucleotide reductase of the extreme thermophile Pyrococcus abyssi is interrupted by three inteins in separate insertion sites. Each intein can facilitate protein splicing when over-expressed in Escherichia coli, with affinity domains serving as the exteins. The influence of the N-terminal flanking residue on the efficiency of splicing is specific to each intein. Each intein has a different downstream nucleophilic residue, and cannot tolerate substitution to a residue of lesser or equal nucleophilicity. The influence of the conserved penultimate His also differs between the inteins.     

蛋白质内含肽及其生物学意义

蛋白质内含肽是存在于前体蛋白质中的一段多肽链,靠自我剪切的方式从前体蛋白中释放出来。蛋白质内含肽的发现,不仅在理论上丰富了遗传信息翻译后加工的内容,而且在实践上有重大的生物学意义,特别是在蛋白质纯化方面有着广泛的应用前景。本文就蛋白质内含肽的发现、特征、鉴定、剪接机制及其生物学意义作一概述。     

Minimization and stabilization of the Mycobacterium tuberculosis recA intein

Many naturally occurring inteins consist of two functionally independent domains, a protein-splicing domain and an endonuclease domain. In a previous study, a 168 amino acid residue mini-intein was generated by removal of the central endonuclease domain of the 440 residue Mycobacterium tuberculosis (Mtu) recA intein. In addition, directed evolution experiments identified a mutation, V67L, that improved the activity of the mini-intein significantly. A recent crystal structure shows that the loop connecting two beta-strands from the N-terminal and C-terminal intein subdomains of the mini-intein is disordered. The goals of the present study were to generate smaller mini-intein derivatives and to understand the basis for reversal of the splicing defect by the V67L mutation. Guided by the structural information, we generated a number of derivatives 135 to 152 residues in length, with V67 or L67. All of the new minimal inteins are functional in splicing. In vivo selection experiments for function showed that by removal of the loop region, 137 residues may be the lower limit for full protein-splicing activity. In addition, the activation effect of the V67L mutation was observed to be universal for mini-inteins longer than 137 residues. Structural and functional analyses indicate that the role of the mutation is in stabilization of the mini-intein core.