ライフサイエンスコーパス: intein

1 cuity of the Int(C) fragment of the GOS-TerL intein.

2 expression domains and fuse each to a split intein.

3 olymers, each containing one half of a split intein.

4 sence of native extein residues flanking the intein.

5 p4 using a modified protein splicing unit or intein.

6 high fitness cost of over 7% for the polB-c intein.

7 in combination with a highly efficient split-intein.

8 ghly efficient Nostoc punctiforme (Npu) DnaE intein.

9 ns also led to inactive gp41-1 and AceL-TerL inteins.

10 ineer promiscuity into other naturally split inteins.

11 efficient trans-splicing and trans-cleaving inteins.

12 engineered versions of naturally split DnaE inteins.

13 ragments, effectively creating two new split inteins.

14 eins and a functional role of HEN domains in inteins.

15 unprecedented rates and yields for all four inteins.

16 eplication, metabolism, and the discovery of inteins.

17 ith a focus on the replication machinery and inteins.

18 sters that relies upon the use of engineered inteins.

19 viral coat proteins; and proteins containing inteins.

20 design of platinum-based compounds targeting inteins.

21 residue is essential for splicing in class 1 inteins.

22 istep posttranslational process catalyzed by inteins.

23 is unique in the structures of thermophilic inteins.

24 s at position one, characteristic of class 1 inteins.

25 in minimized Mycobacterium tuberculosis RecA inteins.

26 et the lateral transfer of mobile introns or inteins.

27 In the Mtu RecA intein, a conserved block-F aspartate (D422) coordinates

28 rally split protein splicing elements called inteins, a novel thiol-hydrazide linker was designed and

29 ca intein has two characteristics of class 3 inteins: a noncontiguous covariant Trp-Cys-Thr triplet a

30 s or previously studied atypical KlbA Ala(1) inteins: (a) cleavage at the N-terminal splice junction

31 tein provides compelling support in favor of inteins acting as pause buttons to arrest protein functi

32 nship between C-extein composition and split intein activity.

33 ordering the intein can profoundly influence intein activity.

34 are no longer bound to the metal atom in the intein adduct.

35 he extein sequences immediately flanking the intein affect splicing and can be defined as the intein

36 tes and/or splicing yields with the Npu DnaE intein after mutation of Cys+1 to Ser+1.

37 frequently used Nostoc punctiforme Npu DnaE intein after the C-extein cysteine nucleophile (Cys+1) w

38 he hyperthermophilic Pyrococcus abyssi PolII intein and extein.

39 ues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a

40 ster than the rate reported for the Npu DnaE intein and gives rise to completed reactions within 20-3

41 This partnership between the intein and its exteins, which implies coevolution of the

42 , which implies coevolution of the parasitic intein and its host protein may provide a novel means of

43 es, and activity may be affected by both the intein and the extein sequence.

44 chemical reaction was able to reactivate the intein and trigger protein splicing.

45 tion in rate compared to the wild-type Cys+1 intein and without mutation of the intein itself to acti

46 We propose an evolutionary model of inteins and a functional role of HEN domains in inteins.

47 ement of intermolecular association of split inteins and could be extended to any interacting protein

48 three-dimensional structure and dynamics of inteins and their biochemical capabilities.

49 6 intein (starting with Ser(1)) is a class 3 intein, and its Ser(1) residue is not required for splic

50 tes this protein splicing from autocatalytic inteins, and may allow the chloroplast to regulate the a

51 se sequence constraints limit the utility of inteins, and thus, a more detailed understanding of thei

52 The fourth describes intein applications that have moved beyond simple techno

53 The split intein approach should be widely applicable for engineer

54 Split inteins are a class of naturally occurring proteins that

55 at their N- and C-terminal splice junctions, inteins are able to excise out of precursor polypeptides

56 Inteins are autoprocessing domains that cut themselves o

57 Curiously, the SufB inteins are found primarily in mycobacterial species tha

58 Inteins are grouped into three classes based on sequence

59 Inteins are invasive genetic elements that self-splice a

60 We challenge the long-considered notion that inteins are merely molecular parasites and posit that so

61 Inteins are mobile genetic elements capable of self-spli

62 Inteins are mobile genetic elements that are spliced out

63 Inteins are mobile genetic elements that self-splice at

64 Inteins are naturally occurring intervening sequences th

65 Inteins are nature's escape artists; they facilitate the

66 Inteins are parasitic genetic elements that excise thems

67 Evolutionary biologists question whether inteins are primordial enzymes or simply selfish element

68 However, natural split inteins are rare and differ greatly in their usefulness

69 The resulting "promiscuous" inteins are shown to be superior reagents for protein cy

70 Inteins are the protein equivalent of introns.

71 ylogenetic analysis, we propose that class 3 inteins arose from a single mutated intein that was spre

72 t into the evolutionary constraints on split intein assembly and should enhance the development of sp

73 This split-intein based method is simpler to implement compared wit

74 We developed a split-intein based split-Cre system for highly efficient Cre-r

75 and efficiency of Cfa should improve current intein based technologies and may provide a platform for

76 Here we present fusion protein- and intein-based fluorescent labeling strategies that can pr

77 Herein, we use intein-based protein semisynthesis to pair a synthetic n

78 Herein, we report an intein-based strategy that allows, for the first time, t

79 or intein engineering and the improvement of intein-based technologies.

80 and should enhance the development of split intein-based technologies.

81 ation for the development and improvement of intein-based tools for chemical biology.

82 tera Gp206 and Nocardioides sp. JS614 TOPRIM inteins belong to based on catalytic mechanism.

83 any of these self-cleaving tags are based on inteins, but other emerging technologies, such as the Fr

84 the expression of either Cre-N-intein-N, or intein-C-Cre-C transgene in different brain regions.

85 ini-intein N159A mutant and an Ssp DnaB mini-intein C1A mutant connected in tandem by a peptide linke

86 the binding of the two partners of the split intein (called protein trans-splicing).

87 nature of the extein substrate bordering the intein can profoundly influence intein activity.

88 we show that when artificially fused, these inteins can be used for rapid generation of protein alph

89 vitro kinetics, we demonstrate that several inteins can catalyze protein trans-splicing in tens of s

90 Several recently identified inteins cannot perform this acyl rearrangement because t

91 However, using intein catalysis to generate peptide sequences exclusive

92 us claims that the +1 extein residue affects intein catalysis.

93 As intein chemistry has widespread use in protein chemistry

94 Split intein chemistry is preceded by efficient molecular reco

95 GFP was fused at its C terminus with an intein; cleavage of the intein provided a unique thioest

96 nal modification where intervening proteins (inteins) cleave themselves from larger precursor protein

97 ent result in engineering a photoactivatable intein compatible with living mammalian cells.

98 Inteins comprise a large family of phylogenetically wide

99 viously identified self-splicing introns and inteins constitute the complex, interconnected mobilome

100 Many inteins contain a homing endonuclease (HEN) domain and r

101 Some inteins contain a homing endonuclease (HEN) responsible

102 ved so far in any of the known or engineered inteins corresponds to the transesterification from an o

103 processing enzymes is closely related to the inteins currently used for protein alpha-thioester gener

104 and dynamics of the minimized and engineered intein DeltaDeltaIhh-V67CM and a single V67L mutant, Del

105 ystal structures of inactive and active mini-inteins derived from inteins residing in the transcripti

106 ini-inteins fused in tandem, termed the dual-intein (DI) domain, to achieve synchronized co-expressio

107 Inteins display a diversity of catalytic mechanisms with

108 due to HEN activity contributes not only to intein dissemination but also to variation at the popula

109 Mediterranean coastline show that the polB-c inteins do not efficiently take over an inteinless popul

110 tion leading to the excision of the internal intein domain from a precursor protein and the concomita

111 The hedgehog-intein domain might also release a subset of toxic nucle

112 ctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequenc

113 Most inteins employ a splicing pathway in which the upstream

114 dynamical aspects of inteins is crucial for intein engineering and the improvement of intein-based t

115 protein scaffold binds to two inactive split intein/enzyme extein protein fragments leading to intein

116 rely molecular parasites and posit that some inteins evolved to regulate host protein function.

117 Inteins excise themselves out of precursor proteins by t

118 ntaneous post-translational process in which inteins excise themselves out of precursor proteins whil

119 a precise self-catalyzed process in which an intein excises itself from a precursor with the concomit

120 yze a protein splicing reaction resulting in intein excision and concatenation of the flanking polype

121 o thioester or oxoester intermediates before intein excision and rearrangement into the new peptide b

122 s spliced efficiently after insertion of the intein flanked by the selected sequences.

123 termediate then collapses into the canonical intein fold.

124 Pab PolII intein has a common HINT (hedgehog intein) fold but contains an extra beta-hairpin that is

125 ve as an environmental sensor, releasing the intein for full activity only at optimal growth conditio

126 rature or solution conditions can unlock the intein for full activity, as can remote extein point mut

127 emission transfer (FRET)-quenched DnaE split inteins for the site-specific labeling and concomitant f

128 n/enzyme extein protein fragments leading to intein fragment complementation, splicing, and activatio

129 al isotopic labeling, we show that one split intein fragment is partly folded, while the other is com

130 arness PTS by rendering association of split intein fragments conditional upon the presence of a user

131 -coils (CCs) to mediate splicing between two intein fragments, effectively creating two new split int

132 of the strong affinity between the two split intein fragments, we devised a streamlined procedure for

133 The FRET assay reports cleavage of the intein from its N-terminal extein.

134 study of a family of inteins, the split DnaE inteins from cyanobacteria.

135 rminal partners of naturally occurring split inteins from three cyanobacteria.

136 Early engineered inteins from various sources allowed the development of

137 The 41 TS inteins function in five different temperature ranges, w

138 hat is based on a pair of self-excising mini-inteins fused in tandem, termed the dual-intein (DI) dom

139 olate the desired protein using a C-terminal intein fusion.

140 reased expression levels relative to other N-intein fusions.

141 on reaction with a complementary synthetic C intein, generated labelled histone.

142 The N- and C-terminal fragments of the four inteins gp41-1, gp41-8, NrdJ-1, and IMPDH-1 were prepare

143 fied from metagenomic databases as the first intein harboring the combination of Ser1 and Cys+1 resid

144 The Pab PolII intein has a common HINT (hedgehog intein) fold but cont

145 NMR structures also show that the Pab PolII intein has a long and disordered loop in place of an end

146 Furthermore, the GOS-TerL intein has an atypical split site close to the N terminu

147 A Thermobifida fusca intein has two characteristics of class 3 inteins: a non

148 The unusual chemistry of inteins has afforded powerful biotechnology tools for co

149 s-splicing (PTS) activity of naturally split inteins has found widespread use in chemical biology and

150 Protein trans-splicing (PTS) by split inteins has found widespread use in chemical biology and

151 in only a few organisms, protein splicing by inteins has since been observed in microorganisms from a

152 14 TOPRIM, and Thermobifida fusca YX Tfu2914 inteins have a mixture of class 1 and class 3 motifs.

153 rvations lead us to the hypothesis that some inteins have adapted to become sensors that play regulat

154 These new inteins have enabled a wide variety of applications in m

155 Naturally split inteins have found widespread use in chemical biology du

156 Split inteins have recently attracted particular interest beca

157 Hedgehog/INTein (HINT) domains catalyzing protein splicing and th

158 Thus, these inteins hold great potential for splicing and cleavage a

159 We report the novel GOS-TerL split intein identified from metagenomic databases as the firs

160 d examining the dispersal efficiency of this intein in a natural, polyploid population.

161 ulated by the native exteins, which lock the intein in an inactive state.

162 The splicing of the intein in the mycobacterial recombinase RecA is specific

163 tions, whereas biotechnologists use modified inteins in an ever increasing variety of applications.

164 We also discuss the incidence of inteins in functionally important sites of their host pr

165 d simple technology development to utilizing inteins in more sophisticated applications, such as bios

166 The discovery of inteins in the early 1990s opened the door to a wide var

167 Synthetic chemists exploit inteins in the semisynthesis of proteins with or without

168 stant residues in the native exteins and the intein, in three-dimensional space.

169 mino acid Int(N) fragment from the AceL-TerL intein, indicating a high degree of promiscuity of the I

170 e persistently found at one specific site in inteins, indicating their potential functional role in p

171 Mycobacteria have inteins inserted into several important genes, including

172 six amino acids that immediately flanked the intein insertion site were randomized.

173 e-defined effects on insertion points of the intein into proteins of interest.

174 mit splicing) and the challenge of inserting inteins into target proteins.

175 The genetically encoded photocaged intein is a general optogenetic tool, allowing effective

176 Therefore, the intein is class 1.

177 ugh this binding process, a whole functional intein is formed resulting in subsequent splicing.

178 spin relaxation indicates that the Pab PolII intein is significantly more rigid than mesophilic intei

179 nature of the recently characterized class 3 inteins is a noncontiguous Trp-Cys-Thr (WCT) motif and t

180 Protein splicing mediated by inteins is a self-processive reaction leading to the exc

181 Despite this fact, the activity of these inteins is context-dependent: certain peptide sequences

182 ding the structural and dynamical aspects of inteins is crucial for intein engineering and the improv

183 undance of powerful applications, the use of inteins is still hampered by limitations in our understa

184 Protein trans-splicing using split inteins is well established as a useful tool for protein

185 lytic reaction where an intervening element (intein) is excised and the remaining two flanking sequen

186 ype Cys+1 intein and without mutation of the intein itself to activate Ser+1 as a nucleophile.

187 BA model by immunization with vWFA2 fused to intein (lacking the GST-tag).

188 coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermoph

189 and point to the intriguing possibility that inteins may act as switches to control extein function.

190 en both domains are expressed does the split intein mediate protein trans-splicing, yielding a full-l

191 Inteins mediate protein splicing, which has found many a

192 dynamic stability might be incompatible with intein-mediated catalysis.

193 The inhibitors were generated by intein-mediated expressed protein ligation using a trunc

194 gh coexpression of the barnase fragments and intein-mediated ligation of the barnase protein fragment

195 Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we

196 st utilization of attenuating +1 mutants for intein-mediated protein manipulations because they facil

197 Intein-mediated protein splicing has found broad biotech

198 Intein-mediated protein splicing raises questions and cr

199 logic, employing hybrid promoters and split intein-mediated protein splicing to integrate signals.

200 Here, we use split-intein-mediated protein transsplicing to reconstitute LT

201 the phospho-S195 Mad2 protein obtained using intein-mediated semisynthesis do not form C-Mad2 on thei

202 of zinc-finger DNA recognition to drive the intein-mediated splicing of an artificial trans-activato

203 etrograde lentiviruses combined with a split-intein-mediated split-Cre-recombinase system in mice to

204 Intein-modified XynB (iXynB) variants were selected that

205 cleavage applications made possible by split intein mutants.

206 The intein N-terminal Cys(1) or Ser(1) residue is essential

207 cers to drive the expression of either Cre-N-intein-N, or intein-C-Cre-C transgene in different brain

208 The DI domain comprises an Ssp DnaE mini-intein N159A mutant and an Ssp DnaB mini-intein C1A muta

209 plored and utilized, it is not clear how the intein navigates through the reaction space.

210 gene encoding DNA polymerase B (polB-c)] and intein-negative cells and examining the dispersal effici

211 moted recombination when intein-positive and intein-negative cells were mated.

212 tween otherwise isogenic intein-positive and intein-negative strains we determined a surprisingly hig

213 between the Npu (fast) and Ssp (slow) split inteins of the DnaE family and find that most impactful

214 rate for the gp41-1 intein, the most active intein on all accounts, was k = 1.8 +/- 0.5 x 10(-1) s(-

215 es were observed when compared with standard inteins or previously studied atypical KlbA Ala(1) intei

216 Inteins (or protein introns) autocatalytically excise th

217 is compatible with several orthogonal split intein pairs, thereby opening the way to the creation of

218 (AFM) was used to directly measure the split intein partner binding at 1 mum/s between native (wild-t

219 process in which a protein editor, called an intein, performs a molecular disappearing act by cutting

220 Split inteins play an important role in modern protein semisyn

221 ese data provide an unprecedented view of an intein poised to carry out the rate-limiting step in pro

222 xamine this question in vivo, by mating polB intein-positive [insertion site c in the gene encoding D

223 f the HEN/intein promoted recombination when intein-positive and intein-negative cells were mated.

224 rough competition between otherwise isogenic intein-positive and intein-negative strains we determine

225 active-site cysteines, Cys1 and Cys+1, in an intein precursor composed of the hyperthermophilic Pyroc

226 rst functional characterization of new split inteins previously identified by bioinformatics from met

227 ucture that can be re-engineered to increase intein promiscuity.

228 The presence of the HEN/intein promoted recombination when intein-positive and i

229 Using a split-intein protein-splicing strategy, we show that a functio

230 Lennon and Belfort introduce inteins - protein introns - and describe how they escape

231 s C terminus with an intein; cleavage of the intein provided a unique thioester moiety at the C termi

232 This comprehensive survey of split inteins provides indispensable information for the devel

233 the genes encoding actin, beta-tubulin, and intein PRP8) revealed that this fungus belongs in the ge

234 The promiscuous inteins reported here thus improve the applicability of

235 Inteins require precise reaction coordination rather tha

236 nactive and active mini-inteins derived from inteins residing in the transcription factor IIB of Meth

237 raphic studies of Pt(II) binding to the RecA intein revealed a complex in which two platinum atoms bi

238 is effect results from predisposition of the intein's catalytic cysteine residues to oxidative and ni

239 ity but recover >60% enzymatic activity upon intein self-splicing at temperatures >59 degrees C.

240 Inteins self-catalytically cleave out of precursor prote

241 Inteins, self-splicing protein elements, interrupt genes

242 on an environmentally sensitive intervening intein sequence is described.

243 ineering approach is presented whereby split intein sequences are flanked by endoplasmic reticulum re

244 te this broad phylogenetic distribution, all inteins share common structural features such as a horse

245 ly of split inteins, the cyanobacterial DnaE inteins, show particular promise, as many of these can s

246 icing, in which an archaeal recombinase RadA intein splices dramatically faster and more accurately w

247 We further demonstrate three-piece intein splicing in mammalian cells and use it to perform

248 Because the intein splicing mechanism is conserved across a range of

249 This unprecedented example of intein splicing stimulation by the substrate of the inva

250 nsitivity of Mycobacterium tuberculosis SufB intein splicing to oxidative and nitrosative stresses wh

251 Residues proximal to the intein-splicing junction in both N- and C-terminal extei

252 The retrograde lentivirus carrying the split-intein-split-Cre system can be applied to study any neur

253 This split-intein-split-Cre system can be used to intersect the exp

254 bines retrograde lentiviruses with the split-intein-split-Cre system in mice to isolate, characterize

255 The mycobacteriophage Catera Gp206 intein (starting with Ser(1)) is a class 3 intein, and i

256 The split intein strategy is able to fully reconstitute the ZF-TFs

257 by reducing the ensemble distribution of the intein structure and by modifying the active sites.

258 l studies also highlight a key region of the intein structure that can be re-engineered to increase i

259 of all possible flanking sequences, studying intein substrate specificity has been difficult.

260 in affect splicing and can be defined as the intein substrate.

261 protein-engineering approaches to show that intein-succinimide formation is critically dependent on

262 n the adjacent backbone amide, leading to an intein-succinimide product and scission of that peptide

263 However, currently used naturally split inteins suffer from an "extein dependence," whereby resi

264 We have generated and characterized 41 intein switches, temperature-sensitive Sce VMA mutations

265 etase/tRNA(CUA) pairs, together with a split intein system were used to biosynthesize a library of ri

266 resent a new part for this function: a split intein T7 RNA polymerase.

267 Split-intein-tagged alpha1C fragments encoding dihydropyridine

268 class 3 inteins arose from a single mutated intein that was spread by phage into predominantly helic

269 an alignment of 73 naturally occurring DnaE inteins that are predicted to be fast.

270 ies as well as the intrinsic features of the inteins that contribute to their genetic mobility.

271 The rate for the gp41-1 intein, the most active intein on all accounts, was k =

272 The most well-characterized family of split inteins, the cyanobacterial DnaE inteins, show particula

273 be the first systematic study of a family of inteins, the split DnaE inteins from cyanobacteria.

274 with a thermostable self-splicing bacterial intein to control the xylanase activity.

275 e protein trans-splicing reaction of a split intein to generate BsAbs without heavy/heavy and light/h

276 e latent catalytic potential of the Npu DnaE intein to splice with an alternative nucleophile and ena

277 quences with Ser+1 that enabled the Npu DnaE intein to splice with only a 5-fold reduction in rate co

278 d the N-terminal fragment of ultrafast split intein to the C terminus of histone H2B, which, on react

279 However, for inteins to effectively contribute to these applications,

280 This has allowed inteins to explore alternative mechanisms with different

281 isms that underlie the remarkable ability of inteins to perform complex sets of choreographed enzymat

282 Mixing these building blocks initiates an intein trans-splicing reaction that yields a hydrogel th

283 Here we solve the crystal structure of an intein trapped in the branched intermediate step in prot

284 This intein-triggered protein hydrogel technology opens new a

285 alternative nucleophile and enables broader intein utility by increasing insertion site choices.

286 ncies and substrate specificity of different inteins vary considerably, reflecting subtle changes in

287 The resulting photocaged intein was inserted into a red fluorescent protein (RFP)

288 Using C1A mutants, all inteins were efficient in the C-terminal cleavage reacti

289 on the evolution and biological function of inteins, whereas the second describes the mechanisms tha

290 nges at the N-terminus and C-terminus of the intein, which are active sites for protein splicing.

291 he hyperthermophilic Pyrococcus abyssi PolII intein, which has a noncanonical C-terminal glutamine in

292 developed around native and engineered split inteins, which allow protein segments expressed separate

293 The discovery of inteins, which are protein-splicing elements, has stimul

294 is significantly more rigid than mesophilic inteins, which may contribute to the higher optimal temp

295 onsensus design approach to engineer a split intein with enhanced stability and activity that make it

296 ng approach that imbues ultrafast DnaE split inteins with minimal extein dependence.

297 sive DNA elements (usually mobile introns or inteins) within the genomes of phage, bacteria, archea,

298 because of the existence of functional mini-inteins without HEN domains.

299 , whereas biochemists seek to understand how inteins work.

300 We show that these intein "zymogens" can be used to create protein sensors