ライフサイエンスコーパス: intein

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

2 in combination with a highly efficient split-intein.

3 ghly efficient Nostoc punctiforme (Npu) DnaE intein.

4 ey are usually made by recombinant fusion to intein.

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

6 expression domains and fuse each to a split intein.

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

8 sence of native extein residues flanking the intein.

9 p4 using a modified protein splicing unit or intein.

10 is harbors a self-splicing element called an intein.

11 hat are then reconstituted by trans-splicing inteins.

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

13 efficient trans-splicing and trans-cleaving inteins.

14 engineered versions of naturally split DnaE inteins.

15 ragments, effectively creating two new split inteins.

16 unprecedented rates and yields for all four inteins.

17 eplication, metabolism, and the discovery of inteins.

18 ith a focus on the replication machinery and inteins.

19 sters that relies upon the use of engineered inteins.

20 viral coat proteins; and proteins containing inteins.

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

22 ineer promiscuity into other naturally split inteins.

23 design of platinum-based compounds targeting inteins.

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

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

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

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

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

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

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

31 he hyperthermophilic Pyrococcus abyssi PolII intein and extein.

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

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

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

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

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

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

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

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

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

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

42 6 intein (starting with Ser(1)) is a class 3 intein, and its Ser(1) residue is not required for splic

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

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

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

46 The split intein approach should be widely applicable for engineer

47 Split inteins are a class of naturally occurring proteins that

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

49 Inteins are autoprocessing domains that cut themselves o

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

51 Inteins are grouped into three classes based on sequence

52 Inteins are invasive genetic elements that self-splice a

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

54 Inteins are mobile genetic elements capable of self-spli

55 Inteins are mobile genetic elements that are spliced out

56 Inteins are mobile genetic elements that self-splice at

57 Inteins are naturally occurring intervening sequences th

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

59 Inteins are parasitic genetic elements that excise thems

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

61 Split inteins are privileged molecular scaffolds for the chemi

62 Inteins are protein segments capable of joining adjacent

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

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

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

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

67 ived from thioredoxin-1 (Trx-1) obtained via intein based expression to enable ligation/desulfurizati

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

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

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

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

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

73 o greatly expanding the applicability of the intein-based protein transsplicing.

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

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

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

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

78 selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional

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

80 Here, we assess the splicing activity of 34 inteins (both uncharacterized and known) using a rapid s

81 They resemble inteins but they operate by a different mechanism that d

82 terminal fragment of the atypically split CL intein by solid-phase peptide synthesis.

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

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

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

86 We show that orthogonal split inteins can be coupled to multiple split transcription f

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

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

89 s fused to protein splicing elements called "inteins" can be separately co-segregated with different

90 However, using intein catalysis to generate peptide sequences exclusive

91 As intein chemistry has widespread use in protein chemistry

92 Split intein chemistry is preceded by efficient molecular reco

93 es of a three-helix bundle protein and split intein chemistry.

94 ineering of a completely cysteine-less split intein (CL intein) that is capable of efficient trans-sp

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

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

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

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

99 Some inteins contain a homing endonuclease (HEN) responsible

100 Lastly, an intein-containing Prp8 precursor model is presented, sug

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

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

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

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

105 Inteins display a diversity of catalytic mechanisms with

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

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

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

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

110 sary for recombination activity; deletion of intein domains or mutation of key intein residues inacti

111 Naturally split inteins drive the ligation of separately expressed polyp

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 functional diphtheria toxin from engineered intein-flanked fragments upon receptor-mediated delivery

124 termediate then collapses into the canonical intein fold.

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 sh a library of 15 mutually orthogonal split inteins for in vivo applications, 10 of which can be sim

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

129 l of an expanded library of orthogonal split inteins for their use in the fields of synthetic biology

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

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

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

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

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

135 Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human funga

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

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

138 Early engineered inteins from various sources allowed the development of

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

140 Although powerful, the intein fusion approach suffers from premature hydrolysis

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

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

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

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

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

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

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

148 ing limitation of all well-established split inteins has been the requirement to carry out the reacti

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

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

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

155 Split inteins have recently attracted particular interest beca

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

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

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

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

160 Working with the Cne Prp8 intein in a reporter assay, we find that the biologicall

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

162 Furthermore, using the CL intein in combination with a nanobody-epitope pair as a

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

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

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

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

167 Inteins in Prp8 are extremely pervasive and are found at

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

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

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

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

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

173 Mycobacteria have inteins inserted into several important genes, including

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

175 The split-intein integrase is a potentially versatile, regulatable

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

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

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

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

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 this process known as protein splicing, the intein itself is not present in the final sequence, thus

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

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

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

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

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

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

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

194 Intein-mediated protein splicing has found broad biotech

195 Intein-mediated protein splicing raises questions and cr

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

197 nsamidation reaction cascade that integrates intein-mediated protein splicing with enzyme-mediated pe

198 Split intein-mediated protein trans-splicing has found extensi

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

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

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

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

203 cleavage applications made possible by split intein mutants.

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

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

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

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

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

209 hat utilizes a naturally split fast splicing intein, Npu.

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

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

212 Inteins (or protein introns) autocatalytically excise th

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

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

215 Split inteins play an important role in modern protein semisyn

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

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

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

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

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

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

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

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

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

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

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

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

228 nstrate a potential application of the split intein-regulated site-specific recombination system in b

229 This demonstrates the potential for split-intein regulation of the forward and reverse reactions u

230 The promiscuous inteins reported here thus improve the applicability of

231 Inteins require precise reaction coordination rather tha

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

233 eletion of intein domains or mutation of key intein residues inactivated recombination.

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

235 We solved the crystal structure of this intein, revealing structural homology among protein spli

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

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

238 Inteins self-catalytically cleave out of precursor prote

239 Inteins, self-splicing protein elements, interrupt genes

240 on an environmentally sensitive intervening intein sequence is described.

241 grase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached

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

243 We have designed a split-intein serine integrase-based system with potential for

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 vant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms.

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

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

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

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

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

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

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

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

265 Split-intein-tagged alpha1C fragments encoding dihydropyridine

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

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

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

269 a completely cysteine-less split intein (CL intein) that is capable of efficient trans-splicing at a

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

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

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

273 The CL intein thus greatly expands the scope of applications fo

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 We used the same split inteins to control the reconstitution of a split Integra

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 tructurally characterize the naturally split intein VidaL.

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

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

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

291 We show that this split intein, which features the shortest known N-terminal fra

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

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

294 are fused to non-interacting pairs of split inteins, which ensure reconstitution of active Cre when

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